Class I Anti-CEA Antibodies and Uses Thereof

ABSTRACT

The present invention provides compositions and methods of use of humanized, chimeric or human Class I anti-CEA antibodies or fragments thereof, preferably comprising the light chain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and the heavy chain variable region CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6). The Class I anti-CEA antibodies or fragments are useful for treating diseases, such as cancer, wherein the diseased cells express CEACAM5 and/or CEACAM6 antigens. The Class I anti-CEA antibodies or fragments are also of use for interfering with specific processes, such as metastasis, invasiveness and/or adhesion of cancer cells, or for enhancing sensitivity of cancer cells to cytotoxic agents and have favorable effects on the survival of subjects with cancer.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/846,062, filed Jul. 29, 2010. The present application claims thebenefit under 35 U.S.C. 119(e) of Provisional U.S. Patent ApplicationSer. No. 61/242,872, filed Sep. 16, 2009, the entire text of which isincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 9, 2010, is namedIMM322US.txt, and is 17,798 bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to compositions for and methods of treatingcancers that express CEACAM 5 (carcinoembryonic antigen, “CEA”) and/orCEACAM6 (NCA-90), such as medullary thyroid cancer (MTC), colorectalcancers, hepatocellular carcinoma, gastric cancer, lung cancer,head-and-neck cancers, bladder cancer, prostate cancer, breast cancer,pancreatic cancer, uterine cancer, ovarian cancer, hematopoieticcancers, leukemia and other cancers in which CEACAM5 and/or CEACAM6 areexpressed. The methods comprise administering a Class I anti-CEAantibody or fragment that targets both CEACAM5 and CEACAM6, preferablyin combination with at least one other therapeutic agent, such asanother antibody, a chemotherapeutic agent, a radioactive agent, anantisense oligonucleotide, an immunomodulator, an immunoconjugate or acombination thereof. The Class I anti-CEA MAb may be administered priorto, with or after administering the therapeutic agent. In preferredembodiments the Class I anti-CEA antibody is a chimeric, humanized orhuman monoclonal antibody (MAb) comprising the light chain variableregion CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2);and QQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDRsequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); andDMGIRWNFDV (SEQ ID NO:6). More preferably, the chimeric, humanized orhuman Class I anti-CEA MAb retains the binding affinity characteristicsand specificities of a parental murine Class I anti-CEA MAb, butpossesses more of the antigenic and effector properties of a humanantibody.

2. Related Art

CEA (CEACAM5) is an oncofetal antigen commonly expressed in a number ofepithelial cancers, most commonly those arising in the colon but also inthe breast, lung, pancreas, thyroid (medullary type) and ovary(Goldenberg et al., J. Natl. Cancer Inst. 57: 11-22, 1976; Shively, etal., Crit. Rev. Oncol. Hematol. 2:355-399, 1985). The human CEA genefamily is composed of 7 known genes belonging to the CEACAM subgroup.These subgroup members are mainly associated with the cell membrane andshow a complex expression pattern in normal and cancerous tissues. TheCEACAM5 gene, also known as CD66e, codes for the CEA protein (Beaucheminet al., Exp Cell Res 252:243, 1999). CEACAM5 was first described in 1965as a gastrointestinal oncofetal antigen (Gold et al., J Exp Med122:467-481, 1965), but is now known to be overexpressed in a majorityof carcinomas, including those of the gastrointestinal tract, therespiratory and genitourinary systems, and breast cancer (Goldenberg etal., J Natl Cancer Inst. 57:11-22, 1976; Shively and Beatty, Crit. RevOncol Hematol 2:355-99, 1985).

CEACAM6 (also called CD66c or NCA-90) is a non-specific cross-reactingglycoprotein antigen that shares some, but not all, antigenicdeterminants with CEACAM5 (Kuroki et al., Biochem Biophys Res Comm182:501-06, 1992). CEACAM6 is expressed on granulocytes and epitheliafrom various organs, and has a broader expression zone in proliferatingcells of hyperplastic colonic polyps and adenomas, compared with normalmucosa, as well as by many human cancers (Scholzel et al., Am J Pathol157:1051-52, 2000; Kuroki et al., Anticancer Res 19:5599-5606, 1999).Relatively high serum levels of CEACAM6 are found in patients with lung,pancreatic, breast, colorectal, and hepatocellular carcinomas. Theamount of CEACAM6 does not correlate with the amount of CEACAM5expressed (Kuroki et al., Anticancer Res 19:5599-5606, 1999).

Expression of CEACAM6 in colorectal cancer correlates inversely withcellular differentiation (Ilantzis et al., Neoplasia 4:151-63, 2002) andis an independent prognostic factor associated with a higher risk ofrelapse (Jantscheff et al., J Clin Oncol 21:3638-46, 2003). Both CEACAM5and CEACAM6 have a role in cell adhesion, invasion and metastasis.CEACAM5 has been shown to be involved in both homophilic (CEA to CEA)and heterophilic (CEA binding to non-CEA molecules) interactions(Bechimol et al., Cell 57:327-34, 1989; Oikawa et al., Biochem BiophysRes Comm 164:39-45, 1989), suggesting to some that it is anintercellular adhesion molecule involved in cancer invasion andmetastasis (Thomas et al., Cancer Lett 92:59-66, 1995). These reactionswere completely inhibited by the Fab′ fragment of an anti-CEACAM5antibody (Oikawa et al., Biochem Biophys Res Comm 164:39-45, 1989).CEACAM6 also exhibits homotypic binding with other members of the CEAfamily and heterotypic interactions with integrin receptors (Stannersand Fuks, In: Cell Adhesion and Communication by the CEA Family,(Stanners ed.) Vol. 5, pp. 57-72, Harwood Academic Publ., Amsterdam,1998). Antibodies that target the N-domain of CEACAM6 interfere withcell-cell interactions (Yamanka et al. Biochem Biophys Res Comm219:842-47, 1996). Many breast, pancreatic, colonic and non-small-celllung cancer (NSCLC) cell lines express CEACAM6 and anti-CEACAM6 antibodyinhibits in vitro migration, invasion, and adhesion of antigen-positivecells (Blumenthal et al, Cancer Res 65:8809-17, 2005).

Anti-CEA antibodies are classified into different categories, dependingon their cross-reactivity with antigens other than CEA. Anti-CEAantibody classification was described by Primus and Goldenberg, U.S.Pat. No. 4,818,709 (incorporated herein by reference from Col. 3, line 5through Col. 26, line 49 of U.S. Pat. No. 4,818,709). The classificationof anti-CEA antibodies is determined by their binding to CEA, meconiumantigen (MA) and nonspecific crossreacting antigen (NCA). Class Ianti-CEA antibodies bind to all three antigens. Class II antibodies bindto MA and CEA, but not to NCA. Class III antibodies bind only to CEA(U.S. Pat. No. 4,818,709). Examples of each class of anti-CEA antibodyare known, such as MN-3, MN-15 and NP-1 (Class I); MN-2, NP-2 and NP-3(Class II); and MN-14 and NP-4 (Class III) (U.S. Pat. No. 4,818,709;Blumenthal et al. BMC Cancer 7:2 (2007)).

The epitopic binding sites of various anti-CEA antibodies have also beenidentified. The MN-15 antibody binds to the A1B1 domain of CEA, the MN-3antibody binds to the N-terminal domain of CEA and the MN-14 antibodybinds to the A3B3 (CD66e) domain of CEA (Blumenthal et al. BMC Cancer7:2 (2007)). There is no direct correlation between epitopic bindingsite and class of anti-CEA antibody. For example, MN-3 and MN-15 areboth Class I anti-CEA antibodies, reactive with NCA, MA and CEA, butbind respectively to the N-terminal and A1B1 domains of CEA. Primus andGoldenberg (U.S. Pat. No. 4,818,709) reported a complicated pattern ofcross-blocking activity between the different anti-CEA antibodies, withNP-1 (Class I) and NP-2 (Class II) cross-blocking binding to CEA of eachother, but neither blocking binding of NP-3 (Class II). However, bydefinition Class I anti-CEA antibodies bind to both CEACAM5 and CEACAM6,while Class III anti-CEA antibodies bind only to CEACAM5.

Anti-CEA antibodies have been suggested for therapeutic treatment of avariety of cancers. For example, medullary thyroid cancer (MTC) confinedto the thyroid gland is generally treated by total thyroidectomy andcentral lymph node dissection. However, disease recurs in approximately50% of these patients. In addition, the prognosis of patients withunresectable disease or distant metastases is poor, less than 30%survive 10 years (Rossi et al., Amer. J. Surgery, 139:554 (1980); Samaanet al., J. Clin. Endocrinol. Metab., 67:801 (1988); Schroder et al.,Cancer, 61:806 (1988)). These patients are left with few therapeuticchoices (Principles and Practice of Oncology, DeVita, Hellman andRosenberg (eds.), New York: JB Lippincott Co., pp. 1333-1435 (1989);Cancer et al., Current Problems Surgery, 22: 1 (1985)). The Class IIIanti-CEA antibody MN-14 has been reported to be effective for therapy ofhuman medullary thyroid carcinoma in an animal xenograft model system,when used in conjunction with pro-apoptotic agents such as DTIC, CPT-11and 5-fluorouracil (U.S. patent application Ser. No. 10/680,734, theExamples section of which is incorporated herein by reference). TheClass III anti-CEA antibody reportedly sensitized cancer cells totherapy with chemotherapeutic agents and the combination of antibody andchemotherapeutic agent was reported to have synergistic effects ontumors compared with either antibody or chemotherapeutic agent alone(U.S. Ser. No. 10/680,734). Anti-CEA antibodies of different classes(such as MN-3, MN-14 and MN-15) have been proposed for use in treating avariety of tumors.

There still exists a need to provide more effective methods of treatingCEA-expressing cancers. The present invention provides compositions andmethods for effective anti-cancer therapy utilizing Class I anti-CEAMAbs, such as chimeric, humanized or human antibodies comprising thelight chain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1);GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chainvariable region CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG(SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6), which are capable ofbinding to both CEACAM5 and CEACAM6. Preferably, the Class I anti-CEAMAb is humanized, and used in combination with a therapeutic agent,particularly a chemotherapeutic agent, to yield an effective therapeutictreatment for CEACAM5- or CEACAM6-expressing cancers with minimaltoxicity. The separate administration of Class I antibody andtherapeutic agent provides enhanced results and the versatility and theflexibility to tailor individual treatment methods.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention concerns a compositioncomprising at least one chimeric, humanized or human Class I anti-CEAMAb or antigen-binding fragment thereof. Where antibody fragments of aClass I anti-CEA antibody are utilized, the fragment may be selectedfrom the group consisting of F(ab′)₂, Fab′, Fab, Fv, scFv fragments andsingle domain antibodies (VHH). Preferably, the Class I anti-CEA MAb isa chimeric, humanized or human monoclonal antibody or fragment thereofthat comprises the light chain variable region CDR sequences SASSRVSYIH(SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); andheavy chain variable region CDR sequences DYYMS (SEQ ID NO:4);FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6). Morepreferably, the variable region sequences of the chimeric, humanized orhuman Class I anti-CEA antibody are attached to human IgG1 or IgG4constant region sequences.

In alternative embodiments, the chimeric, humanized or human Class Ianti-CEA MAb or antigen-binding fragment thereof may be one that blocksor competes for binding to CEACAM5 and/or CEACAM6 with a monoclonalantibody having light chain CDRs comprising CDR1 having an amino acidsequence SASSRVSYIH (SEQ ID NO:1); CDR2 having an amino acid sequenceGTSTLAS (SEQ ID NO:2); and CDR3 having an amino acid sequence QQWSYNPPT(SEQ ID NO:3); and heavy chain CDRs comprising CDR1 having an amino acidsequence DYYMS (SEQ ID NO:4); CDR2 having an amino acid sequenceFIANKANGHTTDYSPSVKG (SEQ ID NO:5); and CDR3 having an amino acidsequence DMGIRWNFDV (SEQ ID NO:6). Such competitive binding or blockingstudies may be performed by any of a wide variety of known bindingassays, as exemplified in FIG. 1 and FIG. 4 and their correspondingExamples.

The skilled artisan will be aware, as discussed in more detail below,that chimeric antibodies retain the framework region (FR) sequences of aparent murine antibody, while humanized and human antibodies willgenerally have human antibody FR sequences. Preferably, the humanizedClass I anti-CEA MAb comprises the heavy chain FR sequences of the humanKOL antibody and the light chain FR sequences of the human REI antibody.However, where appropriate, certain murine FR amino acid residues may besubstituted for corresponding human FR amino acid residues. In preferredembodiments, the substituted murine FR residues may include one or moreamino acid residues selected from heavy chain amino acid residues 28,29, 30, 48 and 49 of SEQ ID NO:10 and light chain amino acid residues21, 47 and 60 of SEQ ID NO:9. In more preferred embodiments, thehumanized Class I anti-CEA MAb comprises the variable region sequencesof SEQ ID NO:7 and SEQ ID NO:8, while the chimeric Class I anti-CEA MAbcomprises the variable region sequences of SEQ ID NO:9 and SEQ ID NO:10.

In various embodiments, the Class I anti-CEA antibody will bind to CEA,MA and NCA and will also bind to human granulocytes. The Class Ianti-CEA MAb binds to both CEACAM5 and CEACAM6. Where the Class Ianti-CEA MAb or fragment thereof is chimeric, humanized, or human, theantibody will preferably retain the binding specificity of a parentalmurine Class I anti-CEA MAb.

In certain embodiments the Class I anti-CEA MAb may be conjugated to atleast one therapeutic agent and/or diagnostic agent to form animmunoconjugate. Such immunoconjugates are of use for deliveringtherapeutic and/or diagnostic agents to a CEACAM5- or CEACAM6-expressingcancer cell and/or for therapy or diagnosis of cancer. In alternativeembodiments, the Class I anti-CEA MAb may be administered as a “naked”(unconjugated) antibody. Either naked antibodies or immunoconjugates maybe administered before, simultaneously with or after another therapeuticanti-cancer agent.

Other embodiments concern methods of diagnosing or treating cancer,comprising administering a Class I anti-CEA antibody or fragment thereofto a subject. For diagnostic purposes, the antibody may be conjugated toat least one diagnostic agent. After allowing the labeled antibody tobind to CEA-expressing cells, the distribution of bound antibody may beimaged or otherwise determined. For treatment of CEA-expressing tumors,the Class I anti-CEA antibody may be conjugated to at least onetherapeutic agent and the immunoconjugate administered to a patient.

In alternative embodiments, the Class I anti-CEA antibody may be part ofa bispecific or multispecific antibody. Such antibodies may contain atleast one binding site for a tumor-associated antigen, such as CEA, andat least one other binding site for a hapten attached to a targetableconstruct. Such bispecific or multispecific antibodies may be used inpretargeting methods for diagnosis or treatment of cancer, as discussedin more detail below. Where pretargeting is used, the bispecific ormultispecific antibody may be administered to a subject and allowed tolocalize to a CEACAM5- or CEACAM6-expressing tumor. A clearing agent mayoptionally be added to enhance clearance of unbound antibody from thecirculation. After allowing a sufficient time for unbound antibody toclear from the circulation, a targetable construct conjugated to atherapeutic and/or diagnostic agent may be administered to the subjectto bind to the antibody localized at the tumor site. Delivery ofdiagnostic agents using a Class I anti-CEA antibody may be performed aspart of an endoscopic, intravascular or intraoperative procedure.

In various embodiments, the therapeutic agent is selected from the groupconsisting of a naked antibody, a cytotoxic agent, a drug, aradionuclide, boron atoms, an immunomodulator, a photoactive therapeuticagent, an immunoconjugate, a hormone, an enzyme, an antisenseoligonucleotide or a combination thereof, optionally formulated in apharmaceutically acceptable vehicle. Preferably, the therapeutic agentis a cytotoxic agent selected from a drug or a toxin. It is contemplatedthat the drug may possess a pharmaceutical property selected from thegroup consisting of antimitotic, alkylating, antimetabolite,antiangiogenic, apoptotic, alkaloid, COX-2, and antibiotic agents andcombinations thereof. Preferably, the drug is selected from the groupconsisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antimetabolites, antibiotics, epipodophyllotoxins, platinum coordinationcomplexes, vinca alkaloids, substituted ureas, methyl hydrazinederivatives, adrenocortical suppressants, antagonists, endostatin,taxols, camptothecins, oxaliplatin, doxorubicins and their analogs.Exemplary oligonucleotides may include siRNA or RNAi molecules. Manyexamples of therapeutic oligonucleotides are known in the art and anysuch known example may be attached to a subject Class I anti-CEAantibody or fragment thereof.

When the therapeutic agent is a microbial, plant or animal toxin, thetoxin can be selected from the group consisting of ricin, abrin, alphatoxin, saporin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin l and Pseudomonas endotoxin.

In another embodiment, an immunomodulator is administered prior to theadministration of a therapeutically effective amount of a Class Ianti-CEA monoclonal antibody or fragment thereof alone or a Class Ianti-CEA monoclonal antibody and at least one therapeutic agent.Immunomodulators may be selected from the group consisting of acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN), a stemcell growth factor, erythropoietin, thrombopoietin and a combinationthereof. Preferably, the lymphotoxin is tumor necrosis factor (TNF), thehematopoietic factor is an interleukin (IL), the colony stimulatingfactor is granulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), the interferon isinterferon-α, -β or -γ, and the stem cell growth factor is designated“S1 factor.”

Included among the cytokines are growth hormones such as human growthhormone, N-methionyl human growth hormone, and bovine growth hormone;parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; prostaglandin, fibroblast growth factor;prolactin; placental lactogen, OB protein; tumor necrosis factor-α and-β; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, IL-25, LW, kit-ligand or FLT-3, angiostatin,thrombospondin, endostatin, tumor necrosis factor and LT. As usedherein, the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines. Administration of a cytokine prior to,simultaneous with, or subsequent to exposure to a cytotoxic agent thatresults in myeloid or hematopoietic toxicity is described in U.S. Pat.No. 5,120,525, the Examples section of which is incorporated herein byreference.

In certain preferred embodiments, the therapeutic agent is aradionuclide that has an energy between 20 and 10,000 keV. Preferably,the radionuclide is selected from the group consisting of ¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.

The methods of the instant invention may further comprise administeringto a subject, either concurrently or sequentially, a therapeuticallyeffective amount of a second humanized, chimeric, human or murinemonoclonal antibody or fragment thereof. The second MAb may bind to atumor-associated antigen (TAA) selected from the group consisting ofcarbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4,CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22,CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a,CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5,CEACAM6, B7, ED-B of fibronectin, Factor H, FHL-1, Flt-3, folatereceptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24,insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R,IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17,IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,MUC3, MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1,EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAILreceptor (R1 and R2), VEGFR, EGFR, PIGF, complement factors C3, C3a,C3b, C5a, C5 and an oncogene product. Similarly, the methods maycomprise administering to a subject, either concurrently orsequentially, a therapeutically effective amount of a second humanized,chimeric or human monoclonal antibody or fragment thereof selected fromthe group consisting of a Class I or Class II or Class III anti-CEAmonoclonal antibody or fragment thereof. Where a second antibody orfragment is administered concurrently with the Class I anti-CEAantibody, it may be administered as a separate molecule or alternativelyas part of a bispecific or multispecific antibody construct with theClass I anti-CEA antibody.

The second MAb may be selected from any of a wide variety of anti-cancerantibodies known in the art, including but not limited to hPAM4 (U.S.Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,151,164), hA19 (U.S. Pat. No.7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No.7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No.7,387,772), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.6,676,924), hRS7 (U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No.7,541,440) and hR1 (U.S. patent application Ser. No. 12/722,645, filedMar. 12, 2010) the Examples section of each cited patent or applicationincorporated herein by reference. The second MAb may also be selectedfrom any anti-hapten antibody known in the art, including but notlimited to h679 (U.S. Pat. No. 7,429,381) and 734 (U.S. Pat. No.7,405,320) or h734 (U.S. Pat. No. 7,405,320), the Examples section ofeach of which is incorporated herein by reference.

In various embodiments, the bispecific or multispecific antibodies orother antibody constructs may be produced as fusion proteins or by useof the Dock-and-Lock (DNL) technology, as described in more detailbelow. Compositions and methods for production and use of DNL constructshave been reported (see, e.g., U.S. Pat. Nos. 7,521,056; 7,527,787;7,534,866; 7,550,143 and 7,666,400 and U.S. patent application Ser. Nos.12/418,877; 12/544,476; 12/731,781; 12/752,649; and 12/754,740, theExamples section of each of which is incorporated herein by reference).DNL complexes are formed by attachment of a selected effector to ananchoring domain (AD) or dimerization and docking domain (DDD) peptidesequence. The DNL complex forms when the DDD sequence spontaneouslydimerizes and binds to the AD sequence. Virtually any effector moietymay be attached to a DDD or AD sequence, including antibodies orantibody fragments, peptides, proteins, enzymes, toxins, therapeuticagents, diagnostic agents, immunomodulators, polymers such aspolyethylene glycol (PEG), cytokines, chemokines, growth factors,hormones and any other type of molecule or complex. In preferredembodiments, the DNL complex may comprise an antibody or fragmentthereof comprising light chain variable region CDR sequences SASSRVSYIH(SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); andheavy chain variable region CDR sequences DYYMS (SEQ ID NO:4);FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6). Inother preferred embodiments, the DNL complex may further comprise acytokine or a polyethylene glycol moiety. These embodiments are notlimiting and the skilled artisan will realize that the claimed DNLcomplexes may comprise a Class I anti-CEA antibody moiety attached tovirtually any other effector moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary comparative CEA binding curve forchimeric MN-15 (cMN-15) vs. the parental murine MN-15 antibody.

FIG. 2 shows a comparison of the heavy chain variable region sequencesof the human KOL antibody (SEQ ID NO:38), the murine MN-15 antibody (SEQID NO:10), and the humanized MN-15 antibody (hMN-15) (SEQ ID NO:8). CDRsequences are boxed and differences between the human KOL and murineMN-15 FR sequences are indicated by underlining in the murine MN-15sequence. Murine FR amino acids that are retained in the hMN-15 sequenceare indicated by underlining in the hMN-15 sequence.

FIG. 3 shows a comparison of the light chain variable region sequencesof the human REI antibody (SEQ ID NO:39), the murine MN-15 antibody (SEQID NO:9), and the humanized MN-15 antibody (hMN-15) (SEQ ID NO:7). CDRsequences are boxed and differences between the human REI and murineMN-15 FR sequences are indicated by underlining in the murine MN-15sequence. Murine FR amino acids that are retained in the hMN-15 sequenceare indicated by underlining in the hMN-15 sequence.

FIG. 4 illustrates an exemplary comparative CEA binding curve forhumanized MN-15 (hMN-15) vs. the parental murine MN-15 antibody.

FIG. 5 shows a schematic structural drawing of the domains of CEACAM5(left) and CEACAM 6 (right). The epitopes recognized by MN-15, MN-3 andMN-14 antibodies on CEACAM5 and CEACAM6 are as noted.

FIG. 6 shows the results of an in vitro endothelial cell adhesion assay.Percentage adhesion of various tumor cells with varying amounts ofexpressed CEACAM5 and CEACAM6 to HUVEC cells in the absence or presenceof MN-15 Fab′, MN-3 Fab′, or Ag8 Fab′ control. Cells were labeled with 1μCi/mL of ³H-thymidine and added to HUVEC cultures and incubated for 30minutes at 37° C. Samples were washed thrice with PBS to removeunattached cells. Attached tumor cells were solubilized with 0.1 N NaOHand radioactivity was measured in a β-scintillation counter. The cpmattached/total cpm added (attaching potential) was determined. Resultsof a typical study are presented. Cell lines used include BT-20(CEACAM5+/CEACAM6+), MCF-7 (CEACAM5−/CEACAM6+), HT-29(CEACAM5−/CEACAM6+), Moser (CEACAM5+/CEACAM6−), MCA38cea(CEACAM5+/CEACAM6−), and MCA38 (CEACAM5−/CEACAM6−). Both MN-15 and MN-3induced a 49% to 58% inhibition in adhesion in four cell lines (P<0.01for MN-3 Fab′ on MCF-7, HT-29, and BT-20; P<0.02 for MN-15 on the samethree lines; and P<0.05 for both Fabs on Moser adhesion to endothelialcells).

FIG. 7 illustrates an exemplary in vivo micrometastasis study. Top,immunohistochemistry of GW-39 tumor sections stained with a nonspecificantibody (A), MN-14 anti-CEACAM5 (B), and MN-15 anti-CEACAM6 (C) andphotographed at 100×. Bottom, survival of nude mice implanted with 30 μLof a 10% suspension of GW-39 human colonic cancer cells. Mice wereimplanted with cells that were preincubated for 30 minutes with MN-3-,MN-15-, or hMN-14-Fab′ (10 μg/mL). Mice also received a single 100 μgdose of the same Fab′ 1 day after cell implantation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods of treatment in which a chimeric,humanized or human Class I anti-CEA antibody or fragment thereof isadministered to a subject with cancer. The methods are of use to treatcancers that express CEACAM5 (CEA) and/or CEACAM6, since Class Ianti-CEA antibodies bind to both antigens. The antibodies may beadministered as naked (unconjugated) antibodies or as immunoconjugatesattached to one or more therapeutic agents. The naked antibodies may beadministered prior to, simultaneously with or after one or more othertherapeutic anti-cancer agents.

The method is useful for treating a wide variety of cancers, includingbut not limited to medullary thyroid carcinoma, colorectal cancers,hepatocellular carcinoma, pancreatic, breast, lung, head-and-neck,bladder, uterine and ovarian cancers, and even cancers that do notexpress CEACAM5 or CEACAM6 at very high levels. For example, treatmentis contemplated in cancers that express CEA at levels of at least 100ng/g of tissue.

As used herein, the phrase “Class I anti-CEA” antibody or antibodyfragment means an antibody or fragment that binds the CEA antigen (orCD66e) and also with normal cross-reactive antigen (NCA), meconiumantigen (MA), granulocytes and CD66a-d (see, Primus et al., U.S. Pat.No. 4,818,709, incorporated herein by reference). In a preferredembodiment, the Class I anti-CEA antibody is a humanized, chimeric orhuman antibody having light chain variable region CDR sequencesSASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ IDNO:3); and heavy chain variable region CDR sequences DYYMS (SEQ IDNO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6).

The mechanism of tumor cell killing by the naked Class I anti-CEAantibody is not known with certainty and likely involves severalmechanisms. It is hypothesized that the naked antibody alone or incombination with a therapeutic agent may affect tumor growth by blockingbiological activities of their respective antigen or by stimulatingnatural immunological functions, such as antibody-dependentcell-mediated cytotoxicity (ADCC) or complement-mediated lysis.Additionally, the naked antibody alone or in combination with thetherapeutic agent may treat and control the cancer by inhibiting cellgrowth and cell cycle progression, inducing apoptosis, inhibitingangiogenesis, inhibiting metastatic activity, and/or affecting tumorcell adhesion. In fact, the anti-CEA antibody or fragment thereof may bemore effective in treating metastases than primary cancers, since themetastases may be more susceptible to antagonists of tumor celladhesion. The present treatment method provides a treatment plan thatmay be optimized to provide the maximum anti-tumor activity forindividual patients by allowing the titration of the antibody and one ormore different therapeutic agents to provide an effective treatmentregimen.

In certain alternative embodiments, a preferred naked murine, chimeric,humanized or human Class I anti-CEA antibody may be a monovalentconstruct, comprising only one binding site for CEACAM5 or CEACAM6. Forexample, a Fab, Fab′ or scFv antibody fragment comprising light chainvariable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ IDNO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDRsequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); andDMGIRWNFDV (SEQ ID NO:6) may be utilized. Such monovalent constructs maybe conjugated to a polymer, such as PEG, to extend its serum half-life,using techniques described in detail below. Another alternativemonovalent antibody fragment may contain a human IgG4 constant regionand hinge. In certain embodiments, the native IgG4 sequence may bemodified by replacing cysteine residues with serine residues, asdiscussed in McDonagh et al., (2006, Protein Eng Des Sel 19:299-307). Instill other alternative embodiments, a monovalent antibody fragment maybe constructed by the DNL technique, also described in detail below.

DEFINITIONS

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, scFv (single chain Fv) and the like. Regardless of structure,an antibody fragment binds with the same antigen that is recognized bythe intact antibody. The term “antibody fragment” includes isolatedfragments consisting of the variable regions, such as the “Fv” fragmentsconsisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavyvariable regions are connected by a peptide linker (“scFv proteins”),and minimal recognition units consisting of the amino acid residues thatmimic the hypervariable region. The Fv fragments may be constructed indifferent ways as to yield multivalent and/or multispecific bindingforms. In the former case of multivalent, they react with more than onebinding site against the CEA epitope, whereas with multispecific forms,more than one epitope (either of CEA or even against CEA and a differentantigen) is bound.

A naked antibody is generally an entire antibody which is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector or immunological functions, such ascomplement fixation and ADCC (antibody dependent cell cytotoxicity).However, the Fc portion may not be required for therapeutic function ofthe antibody, but rather other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,such as inhibition of heterotypic or homotypic adhesion, andinterference in signaling pathways, may come into play and interferewith the disease progression. Naked antibodies include both polyclonaland monoclonal antibodies, and fragments thereof, that include murineantibodies, as well as certain recombinant antibodies, such as chimeric,humanized or human antibodies and fragments thereof. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to any therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, while the constant domains ofthe antibody molecule are derived from those of a human antibody.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule are derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody or antibodyfragment, and is useful in the treatment of a disease. Examples oftherapeutic agents include antibodies, antibody fragments,immunoconjugates, drugs, enzymes, cytotoxic agents, toxins, nucleases,hormones, immunomodulators, chelators, boron compounds, photoactiveagents or dyes, radioisotopes or radionuclides, antisenseoligonucleotides or combinations thereof.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which one or more of the same ordifferent natural antibody, single-chain antibody or antibody fragmentsegments with the same or different specificities are linked. A Class Ianti-CEA fusion protein comprises at least one CEA binding site.Preferably, the Class I anti-CEA fusion protein comprises the lightchain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS(SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain variableregion CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ IDNO:5); and DMGIRWNFDV (SEQ ID NO:6). Valency of the fusion proteinindicates the total number of binding arms or sites the fusion proteinhas to antigen(s) or epitope(s); i.e., monovalent, bivalent, trivalentor mutlivalent. The multivalency of the antibody fusion protein meansthat it can take advantage of multiple interactions in binding to anantigen, thus increasing the avidity of binding to the antigen, or todifferent antigens. Specificity indicates how many different types ofantigen or epitope an antibody fusion protein is able to bind; i.e.,monospecific, bispecific, trispecific, multispecific. Using thesedefinitions, a natural antibody, e.g., an IgG, is bivalent because ithas two binding arms but is monospecific because it binds to one type ofantigen or epitope. In certain embodiments, a fusion protein maycomprise one or more antibodies or fragments thereof linked to adifferent effector protein or peptide, such as a cytokine, hormone,growth factor, binding protein, binding peptide or other effector. Anexemplary fusion protein may comprise an antibody or fragment thereofattached to an AD or DDD peptide, as discussed in detail below.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use acts to stimulate immune cells to proliferate orbecome activated in an immune response cascade, such as macrophages,B-cells, and/or T-cells. An example of an immunomodulator as describedherein is a cytokine, which is a soluble small protein of approximately5-20 kDa that is released by one cell population (e.g., primedT-lymphocytes) on contact with specific antigens, and which acts as anintercellular mediator between cells. As the skilled artisan willunderstand, examples of cytokines include lymphokines, monokines,interleukins, and several related signalling molecules, such as tumornecrosis factor (TNF) and interferons. Chemokines are a subset ofcytokines. Certain interleukins and interferons are examples ofcytokines that stimulate T cell or other immune cell proliferation.

Preparation of Monoclonal Antibodies. Including Chimeric, Humanized andHuman Antibodies

Monoclonal antibodies (MAbs) to specific antigens may be obtained bymethods known to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

MAbs to peptide backbones are generated by well-known methods for Abproduction. For example, injection of an immunogen, such as(peptide)_(N)-KLH, wherein KLH is keyhole limpet hemocyanin, and N=1-30,in complete Freund's adjuvant, followed by two subsequent injections ofthe same immunogen suspended in incomplete Freund's adjuvant intoimmunocompetent animals. The animals are given a final i.v. boost ofantigen, followed by spleen cell harvesting three days later. Harvestedspleen cells are then fused with Sp2/0-Ag14 myeloma cells and culturesupernatants of the resulting clones analyzed for anti-peptidereactivity using a direct binding ELISA. Fine specificity of generatedMAbs can be analyzed for by using peptide fragments of the originalimmunogen. These fragments can be prepared readily using an automatedpeptide synthesizer. For MAb production, enzyme-deficient hybridomas areisolated to enable selection of fused cell lines. This technique alsocan be used to raise antibodies to one or more chelating or other haptenmoieties, such as In(III)-DTPA chelates. Monoclonal mouse antibodies toan In(III)-di-DTPA are known (U.S. Pat. No. 5,256,395 to Barbet).

Another method for producing antibodies is by production in the milk oftransgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63:141-147, 1998; U.S. Pat. No. 5,827,690, both of which are incorporatedin their entirety by reference. Two DNA constructs are prepared whichcontain, respectively, DNA segments encoding paired immunoglobulin heavyand light chains. The DNA segments are cloned into expression vectorsthat contain a promoter sequence that is preferentially expressed inmammary epithelial cells. Examples include, but are not limited to,promoters from rabbit, cow and sheep casein genes, the cowα-lactoglobulin gene, the sheep β-lactoglobulin gene and the mouse wheyacid protein gene. Preferably, the inserted fragment is flanked on its3′ side by cognate genomic sequences from a mammary-specific gene. Thisprovides a polyadenylation site and transcript-stabilizing sequences.The expression cassettes are coinjected into the pronuclei offertilized, mammalian eggs, which are then implanted into the uterus ofa recipient female and allowed to gestate. After birth, the progeny arescreened for the presence of both transgenes by Southern analysis. Inorder for the antibody to be present, both heavy and light chain genesmust be expressed concurrently in the same cell. Milk from transgenicfemales is analyzed for the presence and functionality of the antibodyor antibody fragment using standard immunological methods known in theart. The antibody can be purified from the milk using standard methodsknown in the art.

After the initial raising of antibodies to the immunogen, the variablegenes of the monoclonal antibodies can be cloned from the hybridomacells, sequenced and subsequently prepared by recombinant techniques.General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989). Humanization and chimerization ofmurine antibodies and antibody fragments are well known to those skilledin the art. A chimeric antibody is a recombinant protein that containsthe variable domains including the CDRs derived from one species ofanimal, such as a rodent antibody, while the remainder of the antibodymolecule; i.e., the constant domains, is derived from a human antibody.The use of antibody components derived from humanized and chimericmonoclonal antibodies alleviates potential problems associated with theimmunogenicity of murine constant regions. Techniques for constructingchimeric antibodies are well known to those of skill in the art. As anexample, Leung et al., Hybridoma 13:469 (1994), describe how theyproduced an LL2 chimera by combining DNA sequences encoding the V_(K)and V_(H) domains of the murine LL2 monoclonal antibody, an anti-CD22antibody, with respective human K and IgG₁ constant region domains.

A chimeric monoclonal antibody (MAb) can be humanized by replacing thesequences of the murine FR in the variable domains of the chimeric MAbwith one or more different human FR. As simply transferring mouse CDRsinto human FRs often results in a reduction or even loss of antibodyaffinity, additional modification might be required in order to restorethe original affinity of the murine antibody. This can be accomplishedby the replacement of one or more human residues in the FR regions withtheir murine counterparts to obtain an antibody that possesses goodbinding affinity to its epitope. See, for example, Tempest et al.,Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239: 1534(1988).

Additionally, knowing that chimeric anti-CEA exhibits a binding affinitycomparable to that of its murine counterpart, defective designs, if any,in the original version of the humanized anti-CEA MAb can be identifiedby mixing and matching the light and heavy chains of the chimericanti-CEA to those of the humanized version. Preferably, the humanizedanti-CEA antibody comprises the light chain variable region CDRsequences CDR1 having an amino acid sequence SASSRVSYIH (SEQ ID NO:1);CDR2 having an amino acid sequence GTSTLAS (SEQ ID NO:2); and CDR3having an amino acid sequence QQWSYNPPT (SEQ ID NO:3); and the heavychain variable region CDR sequences CDR1 having an amino acid sequenceDYYMS (SEQ ID NO:4); CDR2 having an amino acid sequenceFIANKANGHTTDYSPSVKG (SEQ ID NO:5); and CDR3 having an amino acidsequence DMGIRWNFDV (SEQ ID NO:6). More preferably, the humanizedanti-CEA antibody comprises the light chain FR sequences of the humanREI antibody and the heavy chain FR sequences of the human KOL antibody.Most preferably, the humanized anti-CEA antibody comprises one or moremurine FR amino acid residues selected from the heavy chain amino acidresidues 28, 29, 30, 48 and 49 of SEQ ID NO:10 and light chain aminoacid residues 21, 47 and 60 of SEQ ID NO:9.

Alternatively, a combination of framework sequences from 2 or moredifferent human antibodies can be used for V_(H) and V_(K) FR sequences.The production of humanized MAbs is described, for example, by Jones etal., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988),Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437(1992), and Singer et al., J. Immun. 150:2844 (1993). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, WO0029584A1.

In another embodiment, a Class I anti-CEA monoclonal antibody is a humanantibody. The human anti-CEA MAb, or another human antibody, can beobtained from a transgenic non-human animal. See, e.g., Mendez et al.,Nature Genetics, 15: 146-156 (1997) and U.S. Pat. No. 5,633,425. Forexample, a human antibody can be recovered from a transgenic mousepossessing human immunoglobulin loci. The mouse humoral immune system ishumanized by inactivating the endogenous immunoglobulin genes andintroducing human immunoglobulin loci. The human immunoglobulin loci areexceedingly complex and comprise a large number of discrete segmentswhich together occupy almost 0.2% of the human genome. To ensure thattransgenic mice are capable of producing adequate repertoires ofantibodies, large portions of human heavy- and light-chain loci must beintroduced into the mouse genome. This is accomplished in a stepwiseprocess beginning with the formation of yeast artificial chromosomes(YACs) containing either human heavy- or light-chain immunoglobulin lociin germline configuration. Since each insert is approximately 1 Mb insize, YAC construction requires homologous recombination of overlappingfragments of the immunoglobulin loci. The two YACs, one containing theheavy-chain loci and one containing the light-chain loci, are introducedseparately into mice via fusion of YAC containing yeast spheroblastswith mouse embryonic stem cells. Embryonic stem cell clones are thenmicroinjected into mouse blastocysts. Resulting chimeric males arescreened for their ability to transmit the YAC through their germlineand are bred with mice deficient in murine antibody production. Breedingthe two transgenic strains, one containing the human heavy-chain lociand the other containing the human light-chain loci, creates progenywhich produce human antibodies in response to immunization.

Unrearranged human immunoglobulin genes also can be introduced intomouse embryonic stem cells via microcell-mediated chromosome transfer(MMCT). See, e.g., Tomizuka et al., Nature Genetics, 16: 133 (1997). Inthis methodology microcells containing human chromosomes are fused withmouse embryonic stem cells. Transferred chromosomes are stably retained,and adult chimeras exhibit proper tissue-specific expression.

As an alternative, human antibody fragments may be isolated from acombinatorial immunoglobulin library. See, e.g., Barbas et al., METHODS:A Companion to Methods in Enzymology 2: 119 (1991), and Winter et al.,Ann. Rev. Immunol. 12: 433 (1994). Many of the difficulties associatedwith generating monoclonal antibodies by B-cell immortalization can beovercome by engineering and expressing antibody fragments in E. coli,using phage display. To ensure the recovery of high affinity, monoclonalantibodies a combinatorial immunoglobulin library must contain a largerepertoire size. A typical strategy utilizes mRNA obtained fromlymphocytes or spleen cells of immunized mice to synthesize cDNA usingreverse transcriptase. The heavy- and light-chain genes are amplifiedseparately by PCR and ligated into phage cloning vectors. Two differentlibraries are produced, one containing the heavy-chain genes and onecontaining the light-chain genes. Phage DNA is isolated from eachlibrary, and the heavy-and light-chain sequences are ligated togetherand packaged to form a combinatorial library. Each phage contains arandom pair of heavy- and light-chain cDNAs and upon infection of E.coli directs the expression of the antibody chains in infected cells. Toidentify an antibody that recognizes the antigen of interest, the phagelibrary is plated, and the antibody molecules present in the plaques aretransferred to filters. The filters are incubated with radioactivelylabeled antigen and then washed to remove excess unbound ligand. Aradioactive spot on the autoradiogramidentifies a plaque that containsan antibody that binds the antigen. Cloning and expression vectors thatare useful for producing a human immunoglobulin phage library can beobtained, for example, from STRATAGENE Cloning Systems (La Jolla,Calif.).

In one embodiment, the antibodies are produced as described in Hansen etal., Cancer, 71:3478 (1993); Hansen et al., U.S. Pat. No. 5,874,540;Primus et al., U.S. Pat. No. 4,818,709, and Shively et al., U.S. Pat.No. 5,081,235, the examples section of each of which is incorporatedherein by reference.

Production of Antibody Fragments

Certain embodiments concern the use of fragments of a Class I anti-CEAantibody, preferably comprising light chain variable region CDRsequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT(SEQ ID NO:3); and heavy chain variable region CDR sequences DYYMS (SEQID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ IDNO:6). Antibody fragments which recognize the same epitope as a parentantibody can be generated by known techniques. For example, antibodyfragments can be prepared by proteolytic hydrolysis of an antibody or byexpression in E. coli of the DNA coding for the fragment. The antibodyfragments are antigen binding portions of an antibody, such as F(ab′)₂,Fab′, Fab, Fv, scFv and the like, and can be obtained by pepsin orpapain digestion of whole antibodies by conventional methods or bygenetic engineering techniques.

For example, an antibody fragment can be produced by enzymatic cleavageof antibodies with pepsin to provide a 100 Kd fragment denoted F(ab′)₂.This fragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 50 Kd Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using papain producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. I, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described in Inbar etal., Proc. Nat'l. Acad. Sci. U.S.A. 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992). Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains which are connected by a peptide linker.These single-chain antigen binding proteins (scFv) are prepared byconstructing a structural gene comprising DNA sequences encoding theV_(H) and V_(L) domains which are connected by an oligonucleotide. Thestructural gene is inserted into an expression vector that issubsequently introduced into a host cell, such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing scFvsare described, for example, by Whitlow et al., Methods: A Companion toMethods in Enzymology, 2:97 (1991). Also see Bird et al., Science242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., BioTechnology 11: 1271 (1993) and Sandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody producing cells. See, for example, Larrick et at., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inacessible to conventional VH-VL pairs. (Muyldermanset al., 2001). Alpaca serum IgG contains about 50% camelid heavychain-only IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may beimmunized with known antigens, such as TNF-α, and VHHs can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca VHH coding sequences have beenidentified and may be used to construct alpaca VHH phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

Humanized, Chimeric and Human anti-CEA Antibodies for Treatment Variousembodiments concern compositions and methods using murine, chimeric,humanized or human Class I anti-CEA antibodies and fragments thereof fortreatment of disease states, such as cancer. Preferably, the Class Ianti-CEA antibody or fragment thereof comprises light chain variableregion CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2);and QQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDRsequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); andDMGIRWNFDV (SEQ ID NO:6). The antibodies can be used to treat any cancerthat expresses CEACAM5 and/or CEACAM6. Exemplary CEACAM5 or CEACAM6expressing carcinomas include without limitation medullary thyroidcancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma,gastric cancer, lung cancer, head-and neck cancers, urinary bladdercancer, prostate cancer, uterine cancer, endometrial cancer, breastcancer, hematopoietic cancer, leukemia and ovarian cancer.

Compositions

A composition as claimed herein may comprise at least one Class Ianti-CEA monoclonal antibody (MAb) or fragment thereof. In certainembodiments, the composition or method of use may also include at leastone therapeutic agent, which may be conjugated to the Class I anti-CEAantibody or not conjugated. In compositions comprising more than oneantibody or antibody fragments, such as a second Class I anti-CEAantibody, the second antibody is non-blocking (i.e., does not blockbinding of the first Class I anti-CEA antibody or antibody fragment toits target antigen).

In one embodiment, the Class I anti-CEA monoclonal antibody or fragmentthereof is humanized, human or chimeric, wherein the humanized, human orchimeric MAb retains substantially the Class I anti-CEA bindingspecificity of a murine Class I anti-CEA MAb. In a preferred embodiment,the Class I anti-CEA monoclonal antibody or fragment thereof comprisesthe light chain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1);GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chainvariable region CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG(SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6).

In a preferred embodiment, the Class I anti-CEA monoclonal antibody orfragment thereof is a humanized antibody that comprises human antibodyFR and constant region sequences, preferably with FR sequences from thehuman REI and KOL antibodies. The framework regions (FRs) preferablycomprise at least one amino acid substituted from the corresponding FRsof a murine monoclonal antibody. Still more preferred, the humanizedantibody or fragment thereof comprises at least one amino acid selectedfrom the group consisting of heavy chain amino acid residues 28, 29, 30,48 and 49 of SEQ ID NO:10 and light chain amino acid residues 21, 47 and60 of SEQ ID NO:9. The amino acid sequence of a preferred humanizedantibody comprises the variable region sequences of SEQ ID NO:7 and SEQID NO:8.

Another embodiment is a composition comprising a chimeric Class Ianti-CEA monoclonal antibody or fragment thereof, optionally with atleast one therapeutic agent, which may be conjugated or unconjugated.Preferably, the chimeric antibody or fragment thereof comprises lightchain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS(SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain variableregion CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ IDNO:5); and DMGIRWNFDV (SEQ ID NO:6). More preferably, the chimericantibody comprises the variable region sequences of SEQ ID NO:9 and SEQID NO:10.

Also described herein is a composition comprising a naked murine,humanized, chimeric or human Class I anti-CEA antibody or fragmentthereof. The composition may optionally comprise a therapeutic agent,such as a second naked or conjugated anti-CEA antibody or antibodyfragment thereof. Where a second anti-CEA antibody is used, it isnon-blocking, i.e., does not block binding of the first Class I anti-CEAantibody or fragment thereof. In other words, both anti-CEA antibodiesor fragments thereof are non-blocking to each other, allowing bothantibodies or fragments thereof to bind to CEA (CD66e).

In still other embodiments, the claimed compositions and methods maycomprise an antibody or fragment that binds to the same epitope ofCEACAM5 or CEACAM6 as a Class I anti-CEA antibody comprising light chainvariable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ IDNO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDRsequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); andDMGIRWNFDV (SEQ ID NO:6). Evidence of epitope binding may be determined,for example, by competitive binding assays that are well known in theart. Additionally, other anti-CEA antibodies, such as Class II or ClassIII anti-CEA antibodies, can be used in combination with the Class Ianti-CEA antibody, in either a naked or conjugated form. For example,one or more chimeric or humanized Class II or Class III anti-CEAantibodies or fragments thereof may be combined with a Class I anti-CEAantibody or fragment thereof.

A number of publications disclose MAbs that recognize CEA and differentmembers of the CEA gene family, such as Thompson et al., J. Clin. Lab.Anal. 5:344 (1991); Kuroki et al., J. Biol. Chem. 266:11810 (1991);Nagel et al., Eur. J. Biochem. 214:27 (1993); Skubitz et al., J.Immunol. 155:5382 (1995); Skubitz et al., J. Leukoc. Biol. 60:106(1996); and Chen et al., Proc. Natl. Acad. Sci. 93: 14851 (1996).

The second antibody or antibody fragment may be either unconjugated(naked) or conjugated to at least one therapeutic agent (immunocougate).Immunoconjugates can be prepared by indirectly conjugating a therapeuticagent to an antibody component. General techniques are described in Shihet al., Int. J. Cancer, 41:832 (1988); Shih et al., Int. J. Cancer,46:1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313. The generalmethod involves reacting an antibody component having an oxidizedcarbohydrate portion with a carrier polymer that has at least one freeamine function and that is loaded with a plurality of drug, toxin,chelator, boron addends, or other therapeutic agents. This reactionresults in an initial Schiff base (imine) linkage, which can bestabilized by reduction to a secondary amine to form the finalconjugate. However, as discussed below many methods of preparingimmunoconjugates are known in the art and any such known method may beused.

Also contemplated are compositions and methods of use comprising ahumanized, chimeric, murine or human Class I anti-CEA antibody orfragment thereof and a second antibody or fragment that binds to adifferent tumor-associated antigen (TAA). In one embodiment, the secondantibody or fragment thereof binds to a TAA selected from the groupconsisting of carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a,CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R,CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70,CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP,PSMA, CEACAM5, CEACAM-6, B7, ED-B of fibronectin, Factor H, FHL-1,Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible factor (HIF),HM1.24, insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β,IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B,MIF, MUC1, MUC2, MUC3, MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia,HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tnantigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α,TRAIL receptor (R1 and R2), VEGFR, EGFR, P1GF, complement factors C3,C3a, C3b, C5a, C5 and an oncogene product.

Methods

Also described herein are methods for treating carcinomas. Exemplarycarcinomas include medullary thyroid carcinoma, colorectal cancer,pancreatic cancer, breast cancer, hepatocellular carcinoma, ovariancancer, gastric cancer, prostate cancer, uterine cancer, hematopoieticcancer, leukemia and various lung, head-and-neck, endometrial, bladder,and liver cancers that express CEACAM5 and/or CEACAM6. The CEA levels inthese types of cancers are lower than present in medullary thyroidcarcinomas but all that is necessary is that the CEACAM5 and/or CEACAM6levels be sufficiently high so that the Class I anti-CEA therapyprovides an effective treatment. Normal colon mucosa has about 100-500ng/gram but carcinomas expressing CEA at levels of about 5 mcg/gram oftissue are suitable for treatment with the methods described herein.

For example, contemplated herein is a method for treating cancercomprising administering to a subject, either concurrently orsequentially, a therapeutically effective amount of a Class I anti-CEAmonoclonal antibody or fragment thereof and at least one therapeuticagent, optionally formulated in a pharmaceutically acceptable vehicle.Preferably, the Class I anti-CEA monoclonal antibody or fragment thereofis chimeric, murine, humanized or human, wherein the Class I anti-CEAMAb retains substantially the Class I anti-CEA binding specificity of aparental murine MAb. More preferably, the Class I anti-CEA antibodycomprises light chain variable region CDR sequences SASSRVSYIH (SEQ IDNO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavychain variable region CDR sequences DYYMS (SEQ ID NO:4);FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6).Preferably the therapeutic agent is a cytotoxic agent, more preferablyan alkylating agent, and most preferably, dacarbazine (DTIC). But inanother embodiment, the therapeutic agent may not be DTIC. Other classesof anti-cancer cytostatic and cytotoxic agents, such as 5-fluorouracil,CPT-11 and oxaliplatin can also be used in combinations with theseantibodies, especially in the therapy of colorectal cancers. In othercancer types, cancer drugs that are known to be effective are also goodcandidates for combining with the antibody therapies proposed herein.

Also contemplated herein is a method for treating CEACAM5 and/orCEACAM6-expressing cancers comprising administering to a subject, eitherconcurrently or sequentially, a therapeutically effective amount of afirst Class I anti-CEA monoclonal antibody or fragment thereof and atleast one therapeutic agent, and a naked or conjugated second humanized,chimeric, human or murine monoclonal antibody or fragment thereof,optionally formulated in a pharmaceutically acceptable vehicle. In oneembodiment, the second antibody or fragment thereof binds to atumor-associated antigen selected from the group consisting human growthhormone, N-methionyl human growth hormone, bovine growth hormone,parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, tumor necrosis factor-α, tumor necrosis factor-β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,erythropoietin (EPO), osteoinductive factors, interferon-α,interferon-β, interferon-γ, macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3,angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.In another embodiment, the second antibody or fragment thereof can be adifferent Class I anti-CEA antibody or fragment thereof that isnon-blocking. Alternatively the second antibody or fragment thereof maybe a Class II or Class III anti-CEA antibody or fragment. The antibodiesand fragments thereof may be administered either concurrently orsequentially with each other or the therapeutic agent.

A naked Class I anti-CEA antibody as described herein can significantlyincrease the chemosensitivity of cancer cells to one or more therapeuticagents. For example, treatment of colon cancer cells with a naked ClassI, anti-CEA antibody comprising light chain variable region CDRsequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT(SEQ ID NO:3); and heavy chain variable region CDR sequences DYYMS (SEQID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ IDNO:6), as described herein, either before or concurrently with atherapeutic agent, such as DTIC, CPT-11,5′-fluorouracil (5-FU) oroxaliplatin, improves a cell's response to a therapeutic agent, such asa cytotoxic drug. Further, these therapeutic methods of treatment with anaked Class I, anti-CEA antibody alone or in combination with atherapeutic agent can be further enhanced by administering animmunomodulator as described herein, prior to the administration of thenaked antibody or the administration of the naked antibody and at leastone of the therapeutic agents.

Multimodal therapies include immunotherapy with a Class I anti-CEAantibody or fragment thereof, and a therapeutic agent, supplemented withadministration of an unconjugated or conjugated antibody, unconjugatedor conjugated fusion protein, or fragment thereof. For example, anunconjugated humanized, chimeric, murine or human Class I anti-CEA MAbor fragment thereof comprising light chain variable region CDR sequencesSASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ IDNO:3); and heavy chain variable region CDR sequences DYYMS (SEQ IDNO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6)may be combined with another naked humanized, murine, chimeric or humanClass I anti-CEA antibody (such as an antibody against a differentepitope on CEA), or a humanized, chimeric, murine or human Class Ianti-CEA antibody immunoconjugate conjugated to a radioisotope,chemotherapeutic agent, cytokine, enzyme, enzyme-inhibitor, hormone orhormone antagonist, metal, toxin, antisense oligonucleotide (e.g.,anti-bcl-2), or a combination thereof.

Preferably, a therapeutic agent of use is a drug used in standard cancerchemotherapy, such as taxane or platinum drugs in ovarian cancer,fluorouracil, CPT-11, and oxaloplatin drugs in colorectal cancer,gemcitabine in pancreatic and other cancers, or taxane derivatives inbreast cancers. COX-2 inhibitors represent still another class of agentsthat show activity in combination with typical cytotoxic agents incancer chemotherapy, and can be used in the same way, but combined inaddition with anti-CEA antibodies alone or in combination with otheranti-TAA antibodies. Optionally, these drugs can be used in combinationwith radiolabeled antibodies, either anti-CEA antibody conjugates orradioimmunoconjugates with other anti-TAA antibodies.

In a preferred embodiment, a naked Class I anti-CEA antibody or fragmentthereof is administered sequentially (either prior to or after) orconcurrently with dacarbazine (DTIC), doxorubin, cyclophosphamide orvincristine, or any combination of these. For example, DTIC andcyclophosphamide may be administered sequentially or concurrently with anaked Class I anti-CEA antibody or fragment thereof. Similarly,5-fluorouracil in combination with folinic acid, alone or in combinationwith irinotecan (CPT-11) or oxaliplatin, is a regimen used to treatcolorectal cancer. Other suitable combination chemotherapeutic regimensare well known, such as with oxaliplatin alone, or in combination withthese other drugs, to those of skill in the art. Accordingly,combination therapy with any of these chemotherapeutic agents and anaked Class I anti-CEA antibody or fragment thereof can be used to treatcancer. In medullary thyroid carcinoma, other chemotherapeutic agentsmay be preferred, such as one of the alkylating agents (e.g., DTIC), aswell as gemcitabine and other more recent classes of cytotoxic drugs.The chemotherapeutic drugs and a naked Class I anti-CEA antibody orfragment thereof, can be administered in any order, or together. In apreferred multimodal therapy, both chemotherapeutic drugs and nakedClass I anti-CEA antibodies or fragments thereof are administeredbefore, after, or co-administered with a conjugated or unconjugatedanti-CEA antibody, fusion protein, or fragment thereof. Preferably, theClass I anti-CEA antibody or fragment thereof is a humanized antibody orfragment thereof.

A preferred treatment schedule of multimodal treatment is administeringboth hMN-15 and DTIC for 3 days, and administering only hMN-15 on days7, 14, 21 and then every 21 days for a treatment duration of 12 months.The doses of hMN-15 are 0.5-15 mg/kg body weight per infusion, morepreferably 2-8, and still more preferably 3-5 mg/kg per infusion, andthe doses of DTIC are as currently applied at the preferred doseclinically, but could also be given at two-thirds or less of the maximumpreferred dose in use, thereby decreasing drug-related adverse events.Repeated drug cycles can be given, such as every 1-6 months, withcontinuation of the naked antibody therapy, or with different schedulesof radiolabeled antibody, drug-conjugated antibody, and inclusion ofcertain cytokines, such as G-CSF and/or GM-CSF, each dose adjusted sothat toxicity to the patient is not enhanced by the therapeuticcombination. The application of a cytokine growth factor, such as G-CSF,may enable even higher doses of myelosuppressive agents, such asradiolabeled antibody or cytotoxic drugs, to be administered, and theseschedules and doses may be adjusted for the patients individually,depending on their disease status and prior therapy, bone marrow statusand tolerability to additional cytotoxic therapies. In a preferredembodiment, the Class I anti-CEA antibody or fragment thereof isadministered in a dosage of 100-600 milligrams protein per dose perinjection. Still more preferred, the Class I anti-CEA antibody orfragment thereof is administered in a dosage of 300-400 milligrams ofprotein per dose per injection, with repeated doses preferred. Thepreferred antibody schedule is infusing once weekly or even lessfrequently, such as once every other week or even every third week,depending on a number of factors, including the extent of the diseaseand the amount of CEA circulating in the patient's blood.

Bispecific and Multispecific Antibodies

In various embodiments, the Class I anti-CEA antibody comprising lightchain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS(SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain variableregion CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ IDNO:5); and DMGIRWNFDV (SEQ ID NO:6) may be incorporated into abispecific or multispecific antibody. Bispecific antibodies are usefulin a number of biomedical applications. For instance, pre-targetingmethods with bispecific antibodies comprising at least one binding sitefor a tumor-associated antigen (TAA), such as CEACAM 5 and/or CEACAM6,as well as at least one binding site for a targetable constructconjugated to therapeutic or diagnostic agents, are also well known inthe art (see, e.g., U.S. Pat. Nos. 7,300,644; 7,138,103; 7,074,405;7,052,872; 6,962,702; 6,458,933, the Examples section of each of whichis incorporated herein by reference). In other embodiments, bispecificantibodies comprising binding moieties targeting two different TAAs, ordifferent epitopes of the same TAA, may be of therapeutic use.

Bispecific antibodies comprising the antigen-binding variable regionsequences of any known anti-TAA antibody may be utilized, including butnot limited to hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No.7,151,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No.7,300,655), hLL1 (U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No.7,074,403), hMu-9 (U.S. Pat. No. 7,387,772), hL243 (U.S. Pat. No.7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hRS7 (U.S. Pat. No.7,238,785), hMN-3 (U.S. Pat. No. 7,541,440) and hR1 (U.S. patentapplication Ser. No. 12/722,645, filed Mar. 12, 2010) the Examplessection of each cited patent or application incorporated herein byreference.

Other antibodies of use may be commercially obtained from a wide varietyof known sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of each ofwhich is incorporated herein by reference. Reduction of the peptidelinker length to less than 12 amino acid residues prevents pairing ofV_(H) and V_(L) domains on the same chain and forces pairing of V_(H)and V_(L) domains with complementary domains on other chains, resultingin the formation of functional multimers. Polypeptide chains of V_(H)and V_(L) domains that are joined with linkers between 3 and 12 aminoacid residues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabodies) andtetramers (termed tetrabodies) are favored, but the exact patterns ofoligomerization appear to depend on the composition as well as theorientation of V-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), inaddition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400 and U.S.patent application Ser. Nos. 12/418,877; 12/544,476; 12/731,781;12/752,649; and 12/754,740, the Examples section of each of which isincorporated herein by reference).

Dock-and-Lock (DNL)

In preferred embodiments, bispecific or multispecific antibodies orother constructs may be produced using the dock-and-lock technology. TheDNL method exploits specific protein/protein interactions that occurbetween the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and the anchoring domain (AD) of A-kinase anchoring proteins(AKAPs) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell. Biol. 2004; 5: 959). PKA, which plays a centralrole in one of the best studied signal transduction pathways triggeredby the binding of the second messenger cAMP to the R subunits, was firstisolated from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol.Chem. 1968; 243:3763). The structure of the holoenzyme consists of twocatalytic subunits held in an inactive form by the R subunits (Taylor,J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found with two typesof R subunits (R1 and RID, and each type has α and β isoforms (Scott,Pharmacol. Ther. 1991; 50:123). The R subunits have been isolated onlyas stable dimers and the dimerization domain has been shown to consistof the first 44 amino-terminal residues (Newlon et al., Nat. Struct.Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to therelease of active catalytic subunits for a broad spectrum ofserine/threonine kinase activities, which are oriented toward selectedsubstrates through the compartmentalization of PKA via its docking withAKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561).

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAP as an excellent pair of linker modules for dockingany two entities, referred to hereafter as A and B, into a noncovalentcomplex, which could be further locked into a stably tethered structurethrough the introduction of cysteine residues into both the DDD and ADat strategic positions to facilitate the formation of disulfide bonds.The general methodology of the “dock-and-lock” approach is as follows.Entity A is constructed by linking a DDD sequence to a precursor of A,resulting in a first component hereafter referred to as a. Because theDDD sequence would effect the spontaneous formation of a dimer, A wouldthus be composed of a₂. Entity B is constructed by linking an ADsequence to a precursor of B, resulting in a second component hereafterreferred to as b. The dimeric motif of DDD contained in a₂ will create adocking site for binding to the AD sequence contained in b, thusfacilitating a ready association of a₂ and b to form a binary, trimericcomplex composed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNLconstructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Pre-Targeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct ortargetable conjugate) that is cleared within minutes from the blood. Apre-targeting Bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are well known in the art, for example, asdisclosed in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J.Nucl. Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987;Oehr et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405;6,962,702; 7,387,772; 7,052,872; 7,138,103; 6,090,381; and 6,472,511.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents. The technique mayalso be utilized for antibody dependent enzyme prodrug therapy (ADEPT)by administering an enzyme conjugated to a targetable construct,followed by a prodrug that is converted into active form by the enzyme.

Therapeutic and Diagnostic Agents

In certain embodiments, the antibodies, antibody fragments or fusionproteins described herein may be administered alone, as a “naked”antibody, fragment or fusion protein. In alternative embodiments, theantibody, fragment or fusion protein may be administered before,concurrently with, or after at least one other therapeutic agent. Inother alternatives, an antibody, fragment or fusion protein may becovalently or non-covalently attached to at least one therapeutic and/ordiagnostic agent to form an immunoconjugate.

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹¹⁰In,¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III). Ultrasound contrast agents may comprise liposomes, suchas gas filled liposomes. Radiopaque diagnostic agents may be selectedfrom barium compounds, gallium compounds and thallium compounds. A widevariety of fluorescent labels are known in the art, including but notlimited to fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.Chemiluminescent labels of use may include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt or anoxalate ester.

Therapeutic agents are preferably selected from the group consisting ofa radionuclide, an immunomodulator, an anti-angiogenic agent, acytokine, a chemokine, a growth factor, a hormone, a drug, a prodrug, anenzyme, an oligonucleotide, a pro-apoptotic agent, a photoactivetherapeutic agent, a cytotoxic agent, which may be a chemotherapeuticagent or a toxin, and a combination thereof. The drugs of use maypossess a pharmaceutical property selected from the group consisting ofantimitotic, antikinase, alkylating, antimetabolite, antibiotic,alkaloid, anti-angiogenic, pro-apoptotic agents and combinationsthereof.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,aplidin, azaribine, anastrozole, anthracyclines, bendamustine,bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), COX-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide,estramustine, epidophyllotoxin, estrogen receptor binding agents,etoposide (VP16), etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide,leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, navelbine, nitrosurea, plicomycin, procarbazine, paclitaxel,pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen,taxol, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa,teniposide, topotecan, uracil mustard, vinorelbine, vinblastine,vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-B; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin and LT. As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

Radioactive isotopes useful for treating diseased tissue include, butare not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.The therapeutic radionuclide preferably has a decay energy in the rangeof 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Augeremitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for analpha emitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV. Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁹⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like. Someuseful diagnostic nuclides may include ¹²⁴I, ¹²³I, ¹³¹I, ¹⁸F, ⁵²Fe,⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or¹¹¹In.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Jori et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Corticosteroid hormones can increase the effectiveness of otherchemotherapy agents, and consequently, they are frequently used incombination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 or minocycline may be of use.

Other useful therapeutic agents comprise oligonucleotides, especiallyantisense oligonucleotides that preferably are directed againstoncogenes and oncogene products of B-cell malignancies, such as bcl-2.Preferred antisense oligonucleotides include those known as siRNA orRNAi.

Immunoconjugates

Any of the antibodies, antibody fragments or antibody fusion proteinsdescribed herein may be conjugated to one or more therapeutic ordiagnostic agents. The therapeutic agents do not need to be the same butcan be different, e.g. a drug and a radioisotope. For example, ¹³¹1 canbe incorporated into a tyrosine of an antibody or fusion protein and adrug attached to an epsilon amino group of a lysine residue. Therapeuticand diagnostic agents also can be attached, for example to reduced SHgroups and/or to carbohydrate side chains. Many methods for makingcovalent or non-covalent conjugates of therapeutic or diagnostic agentswith antibodies or fusion proteins are known in the art and any suchknown method may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein by reference. The general method involvesreacting an antibody component having an oxidized carbohydrate portionwith a carrier polymer that has at least one free amine function. Thisreaction results in an initial Schiff base (imine) linkage, which can bestabilized by reduction to a secondary amine to form the finalconjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, eachincorporated herein by reference. The engineered carbohydrate moiety isused to attach the therapeutic or diagnostic agent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein or to a targetable construct andused to chelate a therapeutic or diagnostic agent, such as aradionuclide. Exemplary chelators include but are not limited to DTPA(such as Mx-DTPA), DOTA, TETA, NETA or NOTA. Methods of conjugation anduse of chelating agents to attach metals or other ligands to proteinsare well known in the art (see, e.g., U.S. Pat. No. 7,563,433, theExamples section of which is incorporated herein by reference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein byreference. Particularly useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used withdiagnostic isotopes in the general energy range of 60 to 4,000 keV, suchas ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc,^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radio-imaging. The same chelates,when complexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI. Macrocyclic chelates such as NOTA, DOTA,and TETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates such as macrocyclic polyethers, which are of interest forstably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F—Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

Pharmaceutically Acceptable Vehicles

The compositions comprising murine, humanized, chimeric or human Class Ianti-CEA MAbs to be delivered to a subject can comprise one or morepharmaceutically acceptable vehicles, one or more additionalingredients, or some combination of these. The Class I anti-CEAantibodies and fragments thereof can be formulated according to knownmethods to prepare pharmaceutically useful compositions. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable vehicle. Other acceptable vehicles are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The Class I anti-CEA antibody or fragment thereof can be formulated forintravenous administration via, for example, bolus injection orcontinuous infusion. Formulations for injection can be presented in unitdosage form, e.g., in ampules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forreconstitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic agent and/or antibody or fragmentthereof. Control release preparations can be prepared through the use ofpolymers to complex or adsorb the antibody. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rateof release of an antibody or fragment thereof from such a matrix dependsupon the molecular weight of the immunoconjugate or antibody, the amountof antibody within the matrix, and the size of dispersed particles.Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al., supra.Other solid dosage forms are described in Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The Class I anti-CEA antibody or fragment thereof may also beadministered to a mammal subcutaneously or by other parenteral routes.Moreover, the administration may be by continuous infusion or by singleor multiple boluses. In general, the dosage of an administered antibodyor fragment thereof for humans will vary depending upon such factors asthe patient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of antibody or fragment thereof that is in therange of from about 0.5 mg/kg to 20 mg/kg as a single intravenousinfusion, although a lower or higher dosage also may be administered ascircumstances dictate. This dosage may be repeated as needed, forexample, once per month for 4-10 months, preferably once per every otherweek for 16 weeks, and more preferably, once per week for 8 weeks. Itmay also be given less frequently, such as every other week for severalmonths or given more frequently and/or over a longer duration. Thedosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

For purposes of therapy, the Class I anti-CEA antibody or fragmentthereof is administered to a mammal in a therapeutically effectiveamount to reduce the size of a tumor as compared to untreated controls.Preferably, the Class I anti CEA antibody or fragment thereof is ahumanized antibody or fragment thereof. A suitable subject for thepresent invention is usually a human, although a non-human mammal oranimal subject is also contemplated. An antibody preparation isadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient mammal.

In particular, an antibody preparation is physiologically significant ifits presence invokes an antitumor response. A physiologicallysignificant effect could also be the evocation of a humoral and/orcellular immune response in the recipient mammal.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one antibody, antibody fragment or fusion protein asdescribed herein. If the composition containing components foradministration is not formulated for delivery via the alimentary canal,such as by oral delivery, a device capable of delivering the kitcomponents through some other route may be included. One type of device,for applications such as parenteral delivery, is a syringe that is usedto inject the composition into the body of a subject. Inhalation devicesmay also be used. In certain embodiments, an Class I anti-CEA antibodyor fragment thereof may be provided in the form of a prefilled syringeor autoinjection pen containing a sterile, liquid formulation orlyophilized preparation of antibody (e.g., Kivitz et al., Clin. Ther.2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person for use of the kit.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an antibody, antibody fragment, fusion protein orbispecific antibody. Exemplary sequences that may be encoded andexpressed include a Class I anti-CEA MAb or fragment thereof, a fusionprotein comprising at least one Class I anti-CEA antibody or fragmentsthereof, a fusion protein comprising at least one first antibody orfragment and at least one second antibody or fragment. The first andsecond antibodies may comprise an Class I anti-CEA antibody and/or anantibody against a tumor or B-cell associated antigen such as B7, CD4,CD5, CD8 CD14, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD70, CD74, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM6, ED-Bfibronectin, IL-2, IL-6, IL-25, MUC1, MUC2, MUC3, MUC4, MIF, NCA-66, Ia,HM1.24, HLA-DR, tenascin, T101, TAC, TRAIL-R1, TRAIL-R2, VEGFR, EGFR,P1GF, ILGF, Flt-3, tenascin, complement factor C5, an oncogene product,Kras, cMET, bcl-2, and/or a hapten on a targetable construct. Inpreferred embodiments, the Class I antibody, antibody fragment, fusionprotein or bispecific antibody comprises the light chain variable regionCDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); andQQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDR sequencesDYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV(SEQ ID NO:6).

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express MAbs in a selected host cell, immunoglobulinenhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci. USA 63: 78-85 (1969).

Also encompassed is a method of expressing antibodies or fragmentsthereof or fusion proteins. The skilled artisan will realize thatmethods of genetically engineering expression constructs and insertioninto host cells to express engineered proteins are well known in the artand a matter of routine experimentation. Host cells and methods ofexpression of cloned antibodies or fragments have been described, forexample, in U.S. Pat. Nos. 7,531,327; 7,537,930 and 7,608,425, theExamples section of each of which is incorporated herein by reference.

General Techniques for Construction of Anti-CEA Antibodies

The V_(κ) (variable light chain) and V_(H) (variable heavy chain)sequences for Class I anti-CEA antibodies may be obtained by a varietyof molecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNAlibrary screening. Specifically, the V genes of a Class I anti-CEA MAbfrom a cell that expresses a murine Class I anti-CEA MAb can beidentified by PCR amplification and DNA sequencing. To confirm theirauthenticity, the cloned V_(L) and V_(H) genes can be expressed in cellculture as a chimeric Ab as described by Orlandi et al., (Proc. Natl.Acad. Sci., USA, 86: 3833 (1989)). Based on the V gene sequences, ahumanized Class I anti-CEA MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine Class I anti-CEA MAb by general molecularcloning techniques (Sambrook et al., Molecular Cloning, A laboratorymanual, 2^(nd) Ed (1989)). The V_(κ) sequence for the MAb may beamplified using the primers VK1BACK and VK1FOR (Orlandi et al., 1989) orthe extended primer set described by Leung et al. (BioTechniques, 15:286 (1993)). The V_(H) sequences can be amplified using the primer pairVH1BACK/VH1FOR (Orlandi et al., 1989) or the primers annealing to theconstant region of murine IgG described by Leung et al. (Hybridoma,13:469 (1994)).

PCR reaction mixtures containing 10 μl of the first strand cDNA product,10 μl of 10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mMMgCl₂, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.),250 μM of each dNTP, 200 nM of the primers, and 5 units of Taq DNApolymerase (Perkin Elmer Cetus) can be subjected to 30 cycles of PCR.Each PCR cycle preferably consists of denaturation at 94° C. for 1 min,annealing at 50° C. for 1.5 min, and polymerization at 72° C. for 1.5min. Amplified V_(κ) and V_(H) fragments can be purified on 2% agarose(BioRad, Richmond, Calif.). The humanized V genes can be constructed bya combination of long oligonucleotide template syntheses and PCRamplification as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

PCR products for V_(κ) can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the V_(κ) PCR products. PCR products for V_(H) canbe subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Individual clones containing the respective PCRproducts may be sequenced by, for example, the method of Sanger et al.(Proc. Natl. Acad. Sci., USA, 74: 5463 (1977)).

Expression cassettes containing the V_(κ) and V_(H) sequences, togetherwith the promoter and signal peptide sequences, can be excised fromVKpBR and VHpBS, respectively, by double restriction digestion asHindIII-BamHI fragments. The V_(κ) and V_(H) expression cassettes can beligated into appropriate expression vectors, such as pKh and pG1g,respectively (Leung et al., Hybridoma, 13:469 (1994)). The expressionvectors can be co-transfected into an appropriate cell, e.g., myelomaSp2/0-Ag14 (ATCC, VA), colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of a chimeric, humanized orhuman. Class I anti-CEA MAb by, for example, an ELISA assay.Alternatively, the V_(κ) and V_(H) expression cassettes can be assembledin the modified staging vectors, VKpBR2 and VHpBS2, excised asXbaI/BamHI and XhoI/BamHI fragments, respectively, and subcloned into asingle expression vector, such as pdHL2, as described by Gilles et al.(J. Immunol. Methods 125:191 (1989) and also shown in Losman et al.,Cancer, 80:2660 (1997)). Another vector that is useful is the GS vector,as described in Barnes et al., Cytotechnology 32:109-123 (2000). Otherappropriate mammalian expression systems are described in Werner et al.,Arzneim.-Forsch./Drug Res. 48(II), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA can becarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992). Following transfection, cells may be grown in 96-wellmicroliter plates in complete HSFM medium (Life Technologies, Inc.,Grand Island, N.Y.) at 37° C., 5% CO₂. The selection process can beinitiated after two days by the addition of hygromycin selection medium(Calbiochem, San Diego, Calif.) at a final concentration of 500 units/mlof hygromycin. Colonies typically emerge 2-3 weeks post-electroporation.The cultures can then be expanded for further analysis. Transfectomaclones that are positive for the secretion of chimeric, humanized orhuman heavy chain can be identified by ELISA assay.

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M glycine buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA and its concentration adjusted to about 1 mg/mlusing PBS. Sodium azide, 0.01% (w/v), is conveniently added to thesample as preservative.

EXAMPLES

The invention is further illustrated by, though in no way limited to,the following examples.

Example 1 Production and Characterization of MN-15 and Other Anti-CEAAntibodies

In 1983, Primus et al. described the first panel of MAbs (NP-1, NP-2,NP-3, and NP-4) that defined NCA-cross-reactive, MA-cross-reactive, andCEA-specific epitopes on the CEA molecule (Primus et al., Cancer Res1983, 43:686-92). NP-1 reacts with NCA (normal cross-reactive antigen),MA (meconium antigen), and CEA (carcinoembryonic antigen) and, in aliquid solution of decreasing ion strength, demonstrates increasingaffinity for CEA, a property shared with unadsorbed goat anti-CEA serumand affinity-purified NCA crossreactive antibody purified from goatanti-CEA serum (Primus et al., 1983). The “ion-sensitive” determinant onCEA first was delineated by Hansen et al. (Clin Res 1971, 19:143) usingunadsorbed anti-CEA polyclonal serum, and subsequently by Haskell et al.(Cancer Res 1983, 43:3857-64) with MAb. NP-2 and NP-3 react with MA andCEA but not with NCA, whereas NP-4 reacts only with CEA (Primus et al.,1983). NP-2 and NP-3 differ because NP-2 binding to CEA is blocked byNP-1, whereas NP-1 does not block binding of NP-3 to CEA. MAb reactivewith NCA, MA, and CEA were designated as Class I MAb; MAb reactive withMA and CEA but unreactive with NCA were designated as Class II MAb; andMAb specific for CEA, being unreactive with NCA and MA, were designatedas Class III MAb (see, e.g., U.S. Pat. No. 4,818,709, the Examplessection of which is incorporated herein by reference). The presentExample describes the production and characterization of a secondgeneration of anti-CEA antibodies, including the MN-15 Class I anti-CEAMAb.

Immunization and Hybridoma Production and Screening

The immunogen used to produce MN-15 and other second generation anti-CEAantibodies was a partially purified CEA preparation derived from theGW-39 human colon adenocarcinoma xenograft (Hansen et al., 1993, Cancer71:3478-85). Tumor was grown in the hind-leg muscle of the goldenhamster, excised, frozen, and stored at −20° C. Homogenization of tumor,extraction with perchloric acid, and concentration of the dialyzedextract by ultrafiltration were performed as described previously(Newman et al., Cancer Res 1974, 34:2125-530). The concentrated extractwas equilibrated by dialysis with saline adjusted to 0.05 M NaH₂PO₄, andchromatographed on a 5.0×90.0 cm G-200 SEPHADEX® gel column. The voidpeak was collected, equilibrated with saline by dialysis, and frozen at−20° C. until used. The concentration of CEA in the extract wasdetermined with the IMMUNOMEDICS® NP-1/NP-3 and NP-1/NP-4 enzymeimmunoassays (EIA) (Immunomedics, Inc., Morris Plains, N.J.).

The following protocol was used to immunize 20-g BALB/c female mice(Harlan, Madison, Wis.), from which the second-generation MN series ofanti-CEA MAb were derived. Mice first were immunized subcutaneously with7.5 μg of CEA in complete Freund adjuvant and then boostedsubcutaneously with 7.5 μg of CEA in incomplete Freund adjuvant on day 3and boosted intravenously with 7.5 μg of CEA in saline on days 6 and 9.On day 20, the first mouse was given 50 μg of CEA intravenously insaline; on day 23 one animal was killed, the spleen cell suspensionswere prepared, and the cells were fused with murine myeloma cellsSP2/0-Ag 14 with the use of polyethylene glycol and then cultured inmedium containing 8-azaguanine.

Hybridoma supernatants were screened for CEA reactive antibody by theROCHE® ¹²⁵I-CEA radioimmunoassay (RIA) in 0.01 and 0.10 M NH₄ acetatebuffer. Positive clones were recloned. The granulocyte reactivity ofCEA-reactive MAb was determined by whole-blood indirect fluorescent flowcytometric analysis (Sharkey et al. Cancer Res., 1990, 50:2823-31). TwoMAb with properties similar to those of NP-2 and NP-3, designated MN-2and MN-6, were selected for additional development. A third MAb, MN-3,reacted with granulocytes but, unlike NP-1 and NP-2, did not demonstrateion sensitivity for CEA binding; it also was selected for development.

The mouse used in the second fusion was immunized on days 1, 3, 6, and9, as described above. It was given 65 μg of CEA in saline intravenouslyon day 278 and 90 μg of CEA in saline on day 404. It was killed on day407. The fusion and screening protocols were the same as those used forthe first mouse. The second fusion yielded one clone (MN-14) that hadproperties consistent with those of NP-4 in that it was unreactive withgranulocytes, bound only 30-40% of ¹²⁵I-CEA in the ROCHE® CEA RIA, anddemonstrated no ion sensitivity of CEA binding in the RIA. A secondclone producing a granulocyte-reactive antibody with propertiesidentical to those of NP-1, MN-15, also was derived from this fusion.

MAb-IgG Purification/Characterization

The MAb were purified from ascites fluid raised in CD2F1 mice (CharlesRiver Laboratories, Wilmington, Mass.) by protein A and ion-exchangecolumn chromatography at 4° C. All of the MAb were of the IgG1 isotypewith kappa light chains, as determined by double gel diffusion.

Enzyme Immunoassay

The reagents, formulation, standards, and assay protocol of the sandwichEIA used in these studies have been described elsewhere (Hansen et al.,Clin Chem 1989, 35:146-51). All assays were performed with microwellscoated with NP-1, except in one experiment, in which microwells werecoated with MN-15. All assays were performed in a sequential mode, withaddition of CEA first, incubation, washing of the wells, and addition ofthe MAb-horseradish peroxidase probe. After the second incubation, theamount of probe bound to CEA was determined by washing out the unboundprobe, adding substrate (o-phenylenediamine/hydrogen peroxide), addingacid to stop the reaction, and quantitating the color product by readingthe absorbance at 490 nm. Horseradish peroxidase was conjugated to MAbNP-4, MN-3, MN-6, and MN-14, as described previously for NP-3 (Hansen etal., Clin Chem 1989, 35:146-51). All conjugates were diluted to matchthe dose-response curve established previously with the NP-3-horseradishperoxidase probe. Probes made with NP-4, MN-3, MN-6, and MN-14 all gavelinear dose-response curves with the GW-39 CEA standard.

MA Standard

Meconium was homogenized in 10 volumes of deionized water and themixture centrifuged at 40,000×g for 30 minutes. The supernatant wasdecanted from the pellet and stored at −20° C. The amount of MA plus CEA(in ng/ml) in the MA standard was determined by assay with the NP-1/NP-3EIA. The amount of CEA (in ng/ml) in the MA standard was determined withthe NP-1/NP-4 assay and found to be 15% of the total MA/CEA activitydetermined with the NP-1/NP-3 EIA.

Blocking Studies

Blocking studies were performed with the same conditions described forquantification of CEA. To assess blocking of binding of the enzymeprobes, microwells were charged with CEA by use of 25 ng/ml of the CEAstandard. Unlabeled MAb were added to MAb-enzyme conjugates, and the EIAassay was completed as described for quantitation of CEA with the NP-3probe.

Tissue Reactivity

Immunoreactivity with blood lymphocytes was determined by live-cellindirect immunofluorescence, with the use of the whole blood stainingmethod developed for use with the ORTHO SPECTRUM® I11 flow cytometer(Ortho Diagnostic Systems, Inc., Raritan, N.J.). Tissue reactivity ofthe MAb was determined by immunohistologic examination performed onfrozen sections and 5 μm sections cut from tissue embedded inParaplast-II. Indirect immunofluorescence on frozen sections wasperformed as previously described, with MAb concentrations of 20 μg/ml(Pawlak-Byczkowska et al., Cancer Res 1989, 49:4568-77) Immunoperoxidasestaining of sections was performed with the avidin-biotin horseradishperoxidase complex method (Vector Laboratories, Burlingame, Calif.)according to the protocol suggested by the manufacturer, with MAbconcentrations of 10 μg/ml.

Animal Studies

For radiolocalization studies, at 4-5 weeks female athymic mice (nu/nu;Harlan, Indianapolis, Ind.) were given subcutaneous injections of 0.2 mlof a 10% suspension of GW-39, prepared from the tumor seriallypropagated in athymic mice. After 2 weeks, the mice were givenintravenous injections of approximately 1 μg of ¹³¹I-labeled NP-4 orMN-14. The animals were killed 7 and 14 days later; the organs wereremoved and radioactivity in the organs determined as describedpreviously (Sharkey et al., Cancer Res 1990, 50:828s-34s).

Results

MAb MN-3 and MN-6 reacted with MA, whereas MN-14 did not detect MA. Allof the MAb-horseradish peroxidase probes were titrated with NP-1-coatedmicrowells charged with 25 ng/ml of CEA to obtain an absorbance of0.6-0.8. Microwells coated with MN-15 yielded results identical to thosefrom microwells coated with NP-1. CEA and MA standards were sent ascoded specimens to commercial laboratories performing the respectivecommercial assays. It was expected that the ROCHE® CEA EIA would bespecific for CEA because the ROCHE® assay uses MAb T86.44, a MAb thatreacts with CEA but not with MA (Neumaier et al., J Immunol 1985,135:3604-09). Both the HYBRITECH® and ABBOTT® commercial CEA EIAdemonstrated cross-reactivity with MA (not shown).

The affinity of radioiodinated MN-14 for CEA was 10 times that of NP-4as determined by adding increasing amounts of CEA to fixed amounts ofMAb and determining free versus bound antibody by high pressure liquidchromatography sizing gel analysis. The 100 ng/ml of radioactiveantibody used in this study to determine the affinity of the MAbapproximates the serum concentration of antibody present in blood afterinfusion of 1 μg of antibody. Between 1 and 2 μg/ml of CEA was requiredto complex 50% of NP-4, with 50% complexation of MN-14 being obtainedwith approximately 0.2 μg/ml of CEA (results not shown).

The normal tissue reactivity of NP-4 and MN-14 was found to beidentical, with both being unreactive with blood granulocytes, spleen,normal liver, and all other normal tissues except the colon (not shown).Staining of normal colon with both MAb was localized to the glycocalyxof the epithelial cells (not shown). Staining of sections of colorectalcarcinoma from 10 different patients and of one lung carcinomademonstrated identical staining patterns with the two MAb, but moreintense staining was observed consistently with MN-14 (not shown).

Results of immunoperoxidase staining of spleen sections with MN-2 andMN-3, performed on frozen sections and sections prepared by conventionaltissue processing (fixed in formaldehyde solution and embedded inParaplast) were determined. The granulocyte antigen reactive with MN-3(spleen sections) was stained in sections fixed in formaldehyde solutionand embedded in Paraplast, whereas the antigen reactive with MN-2 wasnot detected in granulocytes in the section fixed in formaldehydesolution and embedded in Paraplast (not shown). By contrast, MN-2strongly stained CEA present in the section of colon carcinoma fixed informaldehyde solution and embedded in Paraplast (not shown). The patternof MN-2 staining was similar to that observed with MN-3 and MN-14.Although blocking studies cannot prove that two MAb are reactive withthe same epitope, failure to bock provides evidence of reactivity withseparate epitopes.

To evaluate cross-blocking of NP-4 by MN-14, microwells coated with NP-1were incubated with 25 μg of CEA, with reagents described above. Themicrowells then were washed and the NP-4-horseradish peroxidase probeadded, with and without 50 μg/ml of MN-14. After incubation, thesupernatants in the wells were decanted, the wells were washed, and theassay was completed. Binding of the NP-4 probe was inhibited by greaterthan 50%. No blocking of binding of the NP-3 probe was demonstratedunder similar experimental conditions. Blocking of binding of the NP-3probe was evaluated with the same protocol used above for NP-4. BothMN-3 and MN-6 blocked binding of the NP-3 probe to CEA.

NP-1 and MN-15 appeared to have similar properties, demonstrating highgranulocyte reactivity, binding to epitopes of the ion-sensitive CEAdeterminant, and rapidly effecting capture of CEA from solution whencoated on microwells (not shown). NP-2 and MN-2 bind to theion-sensitive determinant but differ from NP-1 in having much lowerreactivity with blood granulocytes and reacting with a granulocyteantigen that was destroyed by conventional tissue processing used inhistologic examination (not shown). MN-3 reacted strongly withgranulocytes but did not react with the ion-sensitive determinant (notshown). It also reacted with CEA bound to NP-1, whereas NP-2demonstrated cross-blocking by NP-1 (not shown). NP-3, MN-6, NP-4, andMN-14 were not reactive with granulocytes or the ion sensitivedeterminant (not shown). NP-3 and MN-6 reacted with MA, whereas NP-4 andMN-14 were not reactive with MA (not shown).

Several tumor localization studies have been performed to compare theeffectiveness of radioiodinated NP-4 with that of MN-14, with superiortargeting being demonstrated for MN-14 in two studies and equivalenttargeting demonstrated in a third study (not shown). Twenty animals wereinjected for each time point for each of the antibodies. As wasobserved, MN-14, as compared with NP-4, provided significantly improvedtumor uptake at both 7 and 14 days after injection (P<0.02 and P<0.001,respectively). The uptake of the two antibodies in normal organs orblood was not significantly different (not shown).

Discussion

MN-14 meets all of the criteria of a Class III anti-CEA MAb, beingunreactive with MA by EIA and not staining normal tissues, with theexception of normal colon. This study adds support to the suggestionthat a lack of reactivity with MA is the single most useful parameterfor selecting Class III anti-CEA MAb. Blocking experiments alone can bemisleading in the classification of CEA MAb, as is apparent from theextensive data published from the International Workshop on epitopereactivity of MAb reactive with CEA (Hammarstrom et al., Cancer Res1989, 49:4852-58). Although blocking experiments placed a number ofClass III MAb into the Gold Group 1 (T84.66, II-16, 11-7, II-10), CEA66, which is strongly reactive with MA and reacts weakly with NCA,cross-blocked CEA binding of a Class III MAb, 11-16. MAb B7.8.5, alsoplaced in Gold Group 1, blocked binding of 11-16 yet reacts with NCA.Thus, the Gold Group 1, constructed on the basis of cross-blocking ofbinding of MAb to CEA, contains Class I, II, and III MAb.

The affinity of MN-14 is approximately 10-fold greater than that ofNP-4, when determined with radioiodinated MAb and identical assayconditions to determine free versus antigen-bound antibody. The affinitydetermined for NP-4 in this study was significantly lower than reportedpreviously with ¹²⁵I-CEA in place of radiolabeled NP-4 (Primus et al.,1983). MN-14, like NP-4 and T86.44, binds less than 50% ofradioiodinated CEA. In the case of T86.44, the low binding of ¹²⁵I hasbeen ascribed to damage of the T86.44-reactive epitope byradioiodination. If radioiodination results in damage to theNP-4-reactive epitope, unlabeled CEA would displace NP-4 moreeffectively, resulting in a falsely elevated affinity constant. For thisreason, we elected to compare the affinity of NP-4 and MN-14 by a directbinding method using radioiodinated antibody, rather than by competitiveblocking of binding of ¹²⁵I-CEA to antibody.

This study also confirms the findings of Bormer et al. (Clin Chem 1991,37:1736-39), that the T84.66 MAb, used as a probe in the commercialROCHE® CEA EIA, is unreactive with MA, whereas commercial ABBOTT® andHYBRITECH® CEA EIA use MAb that react equally with MA and CEA.Experiments in athymic mice bearing human colon cancer xenografts havedemonstrated consistently improved targeting with MN-14 compared withNP-4 (not shown). The results of targeting with NP-4 are highly variablebetween experiments, however; and it has not been possible to concludewhether the superior targeting resulted from increased binding or longerretention of the antibody in the tumor, as suggested by the datapresented herein.

Four of the MN MAb have been evaluated by clinical radioscintigraphystudies. MN-3 strongly targets bone marrow granulocytes and is beinginvestigated for radioimmunodetection of occult infection andinflammation. MN-2 and MN-6 demonstrated similar deficiencies inradioimmunodetection of CEA-containing cancers, as observed for NP-2 andNP-3. ¹³¹I-MN-2 targeted bone marrow, and ¹³¹I-MN-6 accreted in thelumen of the normal colon (unpublished results). Excellent specificityand sensitivity were demonstrated with ¹³¹I-MN-14 in a Phase I clinicalstudy to detect CEA containing tumors by radioimmunodetection, andresults of this study have been described (Sharkey et al., Cancer 1993,71:2082-96).

Conclusions

A second-generation panel of anti-carcinoembryonic antigen (anti-CEA)monoclonal antibodies (MAb) has been generated, and the specificity hasbeen compared with that of the first panel of MAb used to differentiatemeconium antigen (MA) from CEA.

Four of the MAb had similar specificities to the first-generation panelof NP MAb. MN-15, like its first-generation equivalent, NP-1, reactedwith normal cross-reactive antigen (NCA), MA, and CEA. Both MN-15 andNP-1 reacted strongly with granulocytes. MN-2 had properties similar toClass II NP-2, being reactive with MA and CEA, cross-blocking binding toCEA by NP-1, and having low reactivity with granulocytes. Both NP-2 andMN-2 stained granulocytes in frozen tissue sections but showed minimalstaining of granulocytes in sections fixed in formaldehyde solution andembedded in Paraplast. MN-14 demonstrated properties similar to theClass III anti-CEA-specific MAb, NP-4, being unreactive with NCA and MA.MN-14, as compared with NP-4, demonstrated significantly superior tumortargeting in a human colon tumor xenograft model and consistentlystronger staining of frozen sections of colon cancer. A fifth MAb, MN-3,had properties uniquely different from the NP series of MAb, reactingstrongly with granulocytes but not demonstrating the liquid-phaseion-sensitivity binding of CEA exhibited by MN-15 and NP-1.

Example 2 Production of Chimeric and Humanized MN-15 Antibodies

To make a chimeric MN-15 antibody, the murine MN-15 variable regionsequences were attached to human IgG1 constant region sequences, asdescribed in Leung et al., Hybridoma 13:469 (1994). The sequences of themurine MN-15 V_(K) (SEQ ID NO:9) and MN-15 V_(H) (SEQ ID NO:10) used toconstruct the chimeric MN-15 (cMN-15) are shown in FIG. 2 and FIG. 3.FIG. 1 shows the results of a competitive binding study of murine vs.chimeric MN-15, competing with horseradish peroxidase (HRP) labeledmurine MN-15. FIG. 1 demonstrates that the cMN-15 construct has anaffinity for CEA that is virtually identical to the parent murine MN-15antibody, with a dissociation constant in the nanomolar range. Asexpected, the MN-3 (amino terminal) and MN-14 (A3-B3) antibodies, whichbind to different epitopes of CEA than MN-15 (A1B1) did not compete forbinding with HRP-labeled murine MN-15.

To humanize the MN-15 antibody, human variable FRs were selected basedon sequence homology with their murine counterparts. The greatesthomology was found with the human KOL V_(H) and REI V_(K) FRs.Oligonucleotides encoding murine MN-15 CDRs were grafted onto theseheavy and light chain variable region domains, respectively. The MN-15CDR sequences included light chain CDR1 (SASSRVSYIH, SEQ ID NO:1); CDR2(GTSTLAS, SEQ ID NO:2); and CDR3 (QQWSYNPPT, SEQ ID NO:3); and heavychain variable CDR1 (DYYMS, SEQ ID NO:4); CDR2 (FIANKANGHTTDYSPSVKG, SEQID NO:5); and CDR3 (DMGIRWNFDV, SEQ ID NO:6). The humanized variableregion genes were cloned into expression vectors along side human IgG1constant region genes according to Orlandi et al., Proc. Nat'l Acad.Sci. USA 86: 3833 (1989).

Certain murine FR amino acid residues were substituted into the KOL andREI FR region sequences. Specifically, heavy chain amino acid residues28, 29, 30, 48 and 49 and light chain amino acid residues 21, 47 and 60of the murine MN-15 antibody were substituted into the human FRsequences by standard mutagenesis techniques (Sambrook et al., MolecularCloning, A laboratory manual, 2^(nd) Ed (1989)). The resulting hMN-15V_(K) (SEQ ID NO:7) and V_(H) (SEQ ID NO:8) amino acid sequences areshown in FIG. 2 and FIG. 3. The murine MN-15 FR region amino acids thatwere retained in the hMN-15 sequences are indicated by underlining inthe hMN-15 sequences. CDR sequences are bolded and boxed.

FIG. 4 shows a comparison of the binding affinities of murine, chimericand humanized MN-15, in competition with HRP-labeled murine MN-15. Asshown in FIG. 4, the cMN-15 and hMN-15 constructs have virtuallyidentical binding affinities for CEA with the parental murine MN-15antibody, with dissociation constants in the nanomolar range.

Expression vector DNAs may be transfected into SP2/0 by electroporation.Transfectomas secreting the various versions of chimeric or humanizedMN-15 are selected and analyzed. For large-scale production, the cellline is grown in a 16-liter stirred tank bioreactor system(Sulzer-Chemtech, Woodbury, N.Y.). The culture medium is continuouslyharvested from the bioreactor through a 0.2 μm tangential flowmicrofiltration hollow fiber unit. The filtered harvest is collectedcontinuously into a 50-liter reservoir, stored at 4° C., from which themedium stream is pumped to a protein-A column to collect the antibodyfrom the product stream. The harvested antibody is purified by a secondpass through a protein-A column and, finally, Q-Sepharose (Pharmacia,Inc., Piscatway, N.J.). The final product purity is assessed bySDS-PAGE, immunoelectrophoresis, HPLC, and immunoreactivity.

Example 3 Inhibition of Adhesion, Invasion and Metastasis by AntibodiesTargeting CEACAM6 (NCA-90) and CEACAM5 (CEA)

Summary

CEACAM5 and CEACAM6 are overexpressed in many cancers and are associatedwith adhesion and invasion. The effects of three monoclonal antibodiestargeting different epitopes on these antigens (NH₂-terminal [MN-3] andA1B1 domains [MN-15] shared by CEACAM5 and CEACAM6 and the A3B3 domain[MN-14] restricted to CEACAM5) were evaluated in migration, invasion,and adhesion assays in vitro using a panel of human pancreatic, breast,and colonic cancer cell lines, and in the GW-39 human colonicmicrometastasis model in vivo. MN-3 Fab′ and MN-15 Fab′ were botheffective at inhibiting cell migration. MN-15 Fab′ treatment inhibitedinvasion, reducing cell penetration through an extracellular matrix(ECM). MN-3 Fab′ also decreased invasion but was less effective thanMN-15 Fab′ in four of five cell lines. All three monoclonal antibodyFabs decreased adhesion of tumor cells to endothelial cells by 49% to58%. MN-15 Fab′ but not MN-3 or MN-14 Fabs induced a decrease inadhesion of three of six cell lines to the ECM protein, fibronectin, butadhesion to vitronectin, laminin, collagen-I, and collagen-IV was notaffected. In vivo studies showed that treatment with MN-3 Fab′ or MN-15Fab′ of mice implanted with GW-39 human colonic cancer cells increasedtheir survival (P<0.025 and P<0.01, respectively). These studies showthat antibody Fabs that target either CEACAM5 or CEACAM6 affect cellmigration, cell invasion, and cell adhesion in vitro, and that MN-15 andMN-3 Fabs have antimetastatic effects in vivo, resulting in improvedsurvival of mice with metastases. Thus, blocking the N and A1B1 domainsof CEACAM5/CEACAM6 can impede the metastatic process.

Background

The eradication of metastatic disease is crucial for achieving survivalin most patients with cancer. The metastatic process consists of aseries of sequential steps, including invasion of extracellular matrix(ECM), extravasation into vessels, transport in the circulation,adhesion to endothelial cells in a new tissue, extravasation through thevessel wall, and migration and proliferation in response toorgan-specific factors at the new site (Fidler, Cancer Res 1990,50:6130-8). The present Example deals with the development of anantibody-based therapeutic approach to impede metastasis.

Carcinoembryonic antigen (CEA, CEACAM5, and CD66e) is expressed on manycancers (Goldenberg et al., J Natl Cancer Inst 1976, 57:11-22; Gold &Goldenberg, McGill J Med 1997, 3:46-66; Hammarstrom, Semin Cancer Biol1999, 9:67-81) and has been implicated with malignancy, particularlyenhanced metastasis (Yoshioka et al., Jpn J Cancer Res 1998, 89:177-85;Hashino et al., Clin Exp Metastasis 1994, 12:324-28). CEACAM5 functionsas a chemoattractant and as an adhesion molecule (Kim et al., Int JCancer 1999, 82:880-85), and has been reported to promote the metastaticpotential in some experimental tumors (Minami et al., Cancer Res 2001,61:2732-5). CEACAM5 has been shown to be involved in both homophilic(CEACAM5 to CEACAM5) binding between the N domains of antiparallelCEACAM5 molecules on opposite cell surfaces and heterophilic binding(CEACAM5 binding to non-CEACAM5 molecules). Ribozyme-mediatedsuppression of CEACAM5 expression reduces metastases in a nude mousetail vein injection model (Wirth et al., Clin Exp Metastasis 2002,19:155-60), whereas anti-CEACAM5 intact MAb, MN-14, also delayed deathdue to metastasis in a human colorectal cancer model (Blumenthal et al.,Cancer Immuno Immunother 2005, 54:315-27) and reduced growth of the TThuman medullary thyroid cancer xenograft (Stein & Goldenberg, Mol CancerTher 2005, 3:1559-64).

Nonspecific cross-reacting antigen (NCA-90, CEACAM6, and CD66c) is amember of the CEA family that is also expressed on many human cancers(Kuroki et al., Anticancer Res 1999, 19:5599-606). CEACAM6 in tumorscorrelates inversely with cellular differentiation (Ilantzis et al.,Neoplasia 2002, 4:151-63) and is an independent prognostic factorassociated with a higher risk of colorectal cancer relapse (Jantscheffet al., J Clin Oncol 2003, 21:3638-46). CEACAM6 is also an importantprotein associated with cell adhesion; its expression on neutrophils isinvolved in adhesion to cytokine-activated endothelial cells (Kuijperset al., J Cell Biol 1992, 118:457-66). This molecule exhibits homotypicand heterotypic interactions with other members of the CEA family andwith integrin receptors. Antibodies targeting the N domain interferewith cell-cell interactions (Yamanka et al., Biochem Biophys Res Commun1996, 219:842-7). Normal cells routinely undergo apoptosis in theabsence of adhesive interactions with ECM, a phenomenon known as“anoikis” (Frisch et al., J Cell Biol 1994, 124:616-26). Resistance toanoikis, a characteristic of tumor cells, facilitates tumorigenesis andmetastasis. CEACAM5 and CEACAM6 both inhibit anoikis and modulation ofCEACAM6 expression alters the malignant phenotype of cancer cells(Duxbury et al., Oncogene 2004). Silencing the CEACAM6 gene by smallinterfering RNA enhances cell anoikis, increases caspase activation inresponse to anchorage-independent conditions, down-regulates the Aktcell survival pathway, and inhibits metastasis in vivo (Duxbury et al.,Cancer Res 2004, 64:3987-93). Expression of this membrane protein isalso associated with increased invasiveness through a c-src-dependentmechanism (Duxbury et al., Br J Cancer 2004, 91:1384-90). Thus, CEACAM6may represent a therapeutic target to control malignancy and/ormetastasis.

Antigenic sites on CEACAM5 and CEACAM6 have been characterized, andpanels of antibodies recognizing specific epitopes have been generated(Audette et al., Mol Immunol 1987, 24:1177-86; Bjerner et al., TumourBiol 2002, 23:249-62). Three subdomains in the N region that arerequired for intercellular homotypic adhesion have been identified bysite-directed deletions and point mutations (Taheri et al., J Biol Chem2000, 275:26935-43). Binding peptides have been developed to theseregions in an effort to block adhesion (Taheri et al., J Biol Chem 2003,278:14632-9). The concentration of peptide required to block cellaggregation was 1 mg/mL or a 25-fold higher molar concentration than thecomplete antibody, despite the predicted greater penetration of asmaller peptide. This inefficient dose requirement could be due toinstability of the small peptide or due to its low affinity. Wepostulated that using monovalent antibody fragments would be a moreeffective approach for targeting these domains, because stability of theMAb will likely be greater and the affinity higher than correspondingpeptides. Our studies have primarily been done with monovalent fragmentsof the antibodies of interest, rather than intact IgGs, because thesingle binding arm of the Fab will prevent complexation of tumor cells,which might occur with a bivalent IgG. In addition, it allowed us tostudy the efficacy of a CEA/NCA-90-targeted antibody without thepotential effector cell-inducing activity of the Fc region. We haveassessed the expression of CEACAM5 and CEACAM6 on a panel of tumor celllines and have determined the effects of several antibodies targetingdifferent epitopes of CEACAM5 and CEACAM6 for their ability to affecttumor cell migration, invasion, and cell adhesion in vitro. We have alsoevaluated the therapeutic potential of these antibody Fabs incontrolling metastasis and survival of mice bearing aCEACAM5/CEACAM6-expressing human colonic carcinoma.

Materials and Methods

Antibody Production

MN-15 binds to the A1B1 domain (Gold group 4) and MN-3 binds to the Ndomain (Gold group 5) found on both CEACAM5 and CEACAM6, while MN-14binds to the A3B3 domain (Gold group 3) only found on CEACAM5 (FIG. 5).MN-3 and MN-15 were used as murine MAbs, whereas MN-14 was included inits humanized form, hMN-14 or labetuzumab (Sharkey et al., Cancer Res1995, 55:5935-45).

Cell Culture

All cell lines were maintained in monolayer culture having RPMI 1640supplemented with 10% fetal bovine serum (FBS) plus 100 units/mLpenicillin, 100 units/mL streptomycin, 4 mmol/L glutamine, and 1%nonessential amino acids and grown at 37° C. in 95% air and 5% CO₂.Cells were harvested using 1% trypsin and counted by hemocytometer.Viability was determined by trypan blue exclusion.

CEACAM5 and CEACAM6 Expression

Cells were harvested from culture and aliquoted into FACSCAN™ tubescontaining 2 mL Dulbecco's PBS with 0.2% sodium azide and 1% appropriateblocking serum. Cells were incubated with 10 mg/mL of hMN-14anti-CEACAM5 in PBS (+NaN₃+1% blocking serum) for 1 hour on ice,pelleted (1,440 rpm×5 minutes), and supernatant decanted. Some tubeswere then incubated with 10 μg/mL murine MN-15 IgG or MN-3 IgG (todetermine CEACAM6 expression) for 1 hour on ice and pelleted. NeitherMN-15 nor MN-3 bind CEACAM5 once MN-14 is bound so that only CEACAM6 wasdetected by MN-15 or MN-3 in the presence of MN-14. Second antibodyconjugated with FITC (FITC-goat-anti-human for CEACAM5 determination andFITC-goat-anti-mouse for CEACAM6 determination) was added (1:500secondary antibody in PBS+NaN₃+1% blocking serum) for 30 minutes on icein the dark. Cells were pelleted, washed twice with 2 mL ice-coldPBS+NaN₃, and resuspended in 1.5% paraformaldehyde. Fluorescence wasread on a flow cytometer. Percentage of cells that were positive andmean channel fluorescence (MCF) were recorded. CEACAM5 and CEACAM6expression on GW-39 tumor xenografts was determined byimmunohistochemistry using a similar approach of hMN-14 followed bybiotinylated GAH to detect CEACAM5 and preincubation with hMN-14followed by mMN-15 or mMN-3 and biotinylated GAM second antibody todetect CEACAM6 on paraffin sections.

Spontaneous Migration Assay

Glass coverslips were placed in 100×15 mm Petri dishes and UV sterilizedovernight. Suspensions of cancer cells (2−4×10⁵ cells/mL) were preparedfrom 80% to 90% confluent monolayers. Cells were plated on eachcoverslip and incubated for 2 to 5 days to reach 70% to 80% confluence.Two diagonal cell-free paths (“wounds”) were created by dragging asterile yellow pipette tip across the surface. Monolayers were rinsedseveral times to remove floating cells and 4 mL of fresh medium wereadded back in the absence or presence of antibody IgG or Fab′ (10 μg/mL)and incubated at 37° C. After 18 to 24 hours, the medium was removed andcoverslips stained with 1 mL Wright-Giemsa for 1 minute. The stain waswashed off with distilled water, air-dried, and mounted onto slides withCytoseal 60. Repopulation of the wound space was evaluated by countingthe number of cells that migrated into the wound area in 10representative fields. Regions of migration were photographed fordocumentation.

Endothelial Cell Adhesion Assay

Human umbilical vein endothelial cells (HUVEC; Cambrex, San Diego,Calif.) were grown in collagen-coated dishes in EGM Media in ahumidified atmosphere with 5% CO₂ at 37° C. At passages 2 to 5, cellswere plated at a density of 4×10⁴ cells per well in 96-well plates 24hours before the assay. Interleukin-1β (IL-1β, 1 ng/mL) was added 4hours before the assay. At the start of the study, the medium with theIL-1B was removed and fresh DMEM with 1% bovine serum albumin (BSA)added and incubated for 30 minutes. Fresh medium without antibodies orwith 10 μg/mL of MN-15 Fab′, MN-3 Fab′, or Ag8 Fab′ was added. Tumorcells (1×10⁶ cells/mL) prelabeled overnight with ³H-thymidine (100 μLper well using 1.0 μCi/mL) were added to HUVEC cultures and incubatedfor 30 minutes at 37° C. with rotation in medium with 20% FBS. Sampleswere washed thrice with PBS to remove unattached cells. Attached cellswere solubilized with 0.1 N NaOH and radioactivity was measured in a Bscintillation counter. The cpm attached/total cpm added (attachingpotential) was determined.

Adhesion to Extracellular Matrix Proteins Assay

The assay was done using the CYTOMATRIX™ screening kit from CHEMICON®(Kit #ECM205, Temecula, Calif.). The kit contains 96-well plates withstrips coated with fibronectin, vitronectin, laminin, collagen-I, orcollagen-IV. Subconfluent cell cultures were used for these studies.Cells (1×10⁶ cells/mL) were seeded onto coated substrate and incubatedat 37° C. for 1 hour in a CO₂ incubator in PBS containing Ca²⁺/Mg²⁺ (200μL per well). Adherent cells were fixed and stained. The plate waswashed to remove unadhered cells and stained with 100 μL per well of MTS(Cell Titer Aq 96, PROMEGA®, Madison, Wis.) for 5 minutes at roomtemperature. Excess stain was removed with three PBS washes.Solubilization buffer [100 μL of a 50:50 mixture of 0.1 mol/L NaH₂PO₄(pH 4.5) and 50% ethanol] was added to each well. Relative attachmentwas determined using absorbance readings (A_(540 nm)-A_(570 nm)).

Collagen-Based Invasion Assay

The assay was done using CHEMICON® Kit (#ECM551, MILLIPORE®, Billerica,Mass.). Tumor cells at 80% confluence and were serum-starved for 18 to24 hours before the assay was used. Cells were harvested with 5 mL of 2mmol/L EDTA/PBS per 100-mm dish and incubated at 37° C. for 5 to 15minutes. Cells were collected into 10 to 20 mL of quenching medium(serum-free DMEM containing 5% BSA) to inactivate trypsin/EDTA from theharvesting buffer. Cells were pelleted, resuspended in quenching medium(1×10⁶ cells/mL), and appropriate antibody Fabs were added to individualcell aliquots. Cell invasion potential was determined as follows.Baseline invasion and invasion in the presence of a chemoattractant (10%FBS) were measured after a 72-hour incubation period at 37° C. in a 5%CO₂ incubator. The bottom side of the collagen-coated polycarbonatemembrane insert of the invasion chamber was placed in 400 μL of cellstain for 20 minutes at room temperature and washed. The dye wasextracted and transferred into a 96-well microtiter plate forcolorimetric measurements (A_(560 nm)).

In Vivo Therapy Studies

Female athymic nude mice (6 to 8 weeks old) were purchased from TACONIC®(Germantown, N.Y.). Therapy studies were done using ourCEACAM5+/CEACAM6+GW-39 intrapulmonary micrometastasis model, GW-39iv(Sharkey et al., J Natl Cancer Inst 1991, 83:627-32; Blumenthal et al.,Cancer Res 1992, 52:6036-44). The GW-39 human colonic carcinoma has beenmaintained as a serially transplanted signet-ring cell cancer line since1966 (Goldenberg et al., Transplantation 1966, 4:760-4). Stock s.c.GW-39 tumors grown in nude mice were used to prepare a 10% cellsuspension. Cells (30 μL) were injected i.v. into the caudal vein of 5-to 6-week-old female nude mice. This results in ˜50 to 100 tumor nodulesdeveloping in the lungs and a median survival time of 7 to 9 weeks.Cells were pretreated with antibody in vitro (10 μg/mL) beforeimplantation, and then mice were given one additional dose (100 μg permouse i.v.) 1 day after implantation. Body weight was monitored weeklyand animal survival recorded. Results were analyzed by the Kaplan-Meierestimated survival curves, and significance was determined with thelog-rank comparison of survival curves. Median survival time for eachtreatment group also was determined. All studies used 10 mice pertreatment group.

Results

CEACAM5 and CEACAM6 Expression in Cell Lines

Flow cytometric analysis of CEACAM5 and CEACAM6 expression in a panel of31 to 33 commonly used solid tumor cell lines revealed that only 6 of 29(20.7%) expressed significant amounts of CEACAM5, whereas 16 of 30(53.3%) lines were positive for CEACAM6 (Table 1). Two cancer cell lineswere CEACAM5+/CEACAM6− (Moser and LNCAP), four were CEACAM5+/CEACAM6+,and 12 were CEACAM5−/CEANCAM6+. The CEACAM6+ cell lines included 7 of 10breast cancers, one of four ovarian cancers, three of four coloncancers, three of four pancreatic cancers, one of six prostate cancers,and two of four non-small cell lung cancer lines. Many of these tumorlines had >95% of cells expressing the CEACAM6 antigen and, in somecases, expression was very high (MCF of 641 for ZR75-30, 702 for BXPC3,and 476 for CaPAN-1). In contrast, the most positive CEACAM5-expressinglines had <60% of cells expressing the antigen, with a MCF<120.

TABLE 1 Number of Tissue Cores Analyzed for Each Histotype. TumorHistotype N Tumor Histotype N Breast Infiltrating Ductal 30 ColonAdenocarcinoma 41 Papillary 8 Pancreas Well Diff 1 Lobular 4 Well-ModDiff 16 Phyllodes 4 Mod Diff 2 Lung Well Diff Adeno 5 Mod-Poorly Diff 6Mod Diff Adeno 5 Poorly Diff 1 Poorly Diff Adeno 5 Ovary Serous Adeno 5Well Diff Squamous 5 Mucinous Adeno 4 Mod Diff Squamous 5 Clear Cell 5Poorly Diif Squamous 5 Transitional Cell 5 Bronchioalveolar 3Endometroid 4 Large-Cell 2 Brenner 4 Neuroendocrine Yolk Sac 3Large-Cell 3 Granulosa 3 Small-Cell 2 Dysgerminoma 3 Prostate Stage II21 Stage III 15 Stage IV 4

Effect of Anti-CEACAM5 and Anti-CEACAM6 Antibodies on Cell Migration

Using the wound-healing assay, we assessed cell migratory activity invitro in a panel of cell lines. LS174T and HT-29 cells showed the mostmigratory activity, whereas ZR75-30, MCF-7, and BXPC3 cells showed verylittle migration (results not shown). Compared with an irrelevantantibody, MN-3 and MN-15 intact IgG and Fab′ all reduced migration inboth cell lines. In HT-29, the number of migrating cells per fielddecreased from 14.9±9.1 to 6.1±0.9 and 3.7±1.6 cells with MN-3 IgG andFab′, respectively, and to 5.5±1.8 and 5.4±0.8 with MN-15 IgG and Fab′,respectively (P<0.01 for all comparisons of MAb treatment with untreatedcells; n=10 for each arm). In LS174T studies, untreated cells had38.8±10.9 migrating cells per field, whereas cells treated with MN-3 IgGor Fab′ had 21.4±5.2 and 18.1±1.6 migrating cells, respectively (P<0.001versus untreated), and cells treated with MN-15 IgG or Fab′ had 20.3±0.1and 21.6±1.2 migrating cells, respectively (P<0.001 versus untreated).Thus, blocking either the N or A1B1 domain of CEACAM6 with either anintact IgG or a monovalent Fab inhibited migration in vitro.

Effect of MN-15 and MN-3 on Tumor Cell Adhesion to Endothelial Cells

Because both CEACAM5 and CEACAM6 are known to have an adhesion role, weevaluated whether blocking these antigens reduces adhesion toendothelial cells (FIG. 6). In the CEACAM5−/CEACAM6− MCA38 murinecolonic cancer line, neither MN-3 Fab′ targeting the N domain nor MN-15Fab′ targeting the A1B1 domain of CEACAM6 had an effect on tumorcell-endothelial cell adhesion (not shown). In contrast, both antibodiesreduced endothelial cell binding of MCA38cea (a human CEA-transfectedline) from 11.68±0.77% to 6.42±2.1% (MN-3) and 5.53±1.15% (MN-15), beingsignificant (P<0.05) for both Fabs (FIG. 6). Both antibodies induced a49% to 58% adhesion-inhibition in four other cell lines (P<0.01 for MN-3Fab′ on MCF-7, HT-29, and BT-20; P<0.02 for MN-15 on the same threelines; and P<0.05 for both Fabs on Moser cell adhesion to endothelialcells, FIG. 6). An isotype-matched irrelevant antibody Fab′ (Ag8) didnot affect tumor cell-endothelial cell adhesion nor did the MN-14anti-CEA Fab′. The magnitude of the anti-adhesion effect did not seemstrictly correlated with the amount of CEACAM5 or CEACAM6 expressed,because MN-15 Fab′ resulted in a greater decrease in adhesion in HT-29cells (48%) than with MCF-7 cells (41%) that express much more CEACAM6.

Effect of Anti-CEACAM5 and Anti-CEACAM6 Antibodies on Tumor CellAdhesion to Extracellular Matrix Proteins

In addition to tumor cell binding to endothelial cells, these cells canalso bind ECM proteins. The extent of tumor cell binding to ECM proteinsvaries among different cell lines. MCF-7 bound well to four of fiveproteins (A₅₆₀>1.1) except laminin (A_(560 nm)=0.2±0.04), whereasZR75-30 attached weakly to all five ECM proteins evaluated(A_(560 nm)<0.45). MDA-468 bound quite well to collagen-I andcollagen-IV (A_(560 nm)=1.25±0.07 and 0.97±0.03, respectively) but notas well to fibronectin, vitronectin, or laminin. The reverse pattern wasseen with CaPAN-1 (A_(560 nm)=1.78±0.21 for fibronectin, 0.88±0.11 forvitronectin, 1.14±0.09 for laminin, 0.07±0.00 for collagen-I, and0.13±0.08 for collagen-IV). None of the antibodies (IgG or Fab′)modulated adhesion to vitronectin, laminin, collagen-I, or collagen-IV(results not shown). However, MN-15 IgG decreased adhesion of MCF-7,MDA-468, and CaPAN-1 to fibronectin by 29% (P<0.01), 51% (P<0.001), and47% (P<0.02), respectively, while not affecting the binding of ZR75-30,BXPC3, or Moser to fibronectin. Two of these three MAb-unresponsive celllines had the lowest baseline adhesion to fibronectin.

Effect of MN-3, MN-15, and MN-14 Fabs on Tumor Cell Invasion

Specific invasion in response to FBS as a chemoattractant was 1.9-fold(LS174T) to 7.4-fold (BXPC3) higher than in the absence of FBS (notshown). MN-15 Fab′ was more effective than MN-3 Fab′ at reducing tumorcell invasion in vitro in five cell lines that expressed CEACAM5,CEACAM6, or both antigens. MN-14 anti-CEA (CEACAM5) had no effect ontumor cell invasion in most cell lines. MDA-231 expresses neitherantigen, and its invasion was not reduced by either MN-3 or MN-15 IgG orFab′. For the five antigen-positive lines, both the intact IgG and themonovalent Fab′ for a given antibody were equally effective. Forexample, MN-15 Fab′ reduced cell invasion of LS174T, MCF-7, GR75-30,BXPC3, and CaPAN-1 cells by 30% (P<0.02), 77% (P<0.01), 49% (P<0.01),44% (P<0.01), and 73% (P<0.002), respectively. The effect of MN-3 Fab′on the same five lines was a reduction in invasion of 3% (P═NS), 47%(P<0.01), 59% (P<0.01), 0% (P═NS), and 55% (P<0.05), respectively. Thus,the A1B1 domain of CEACAM6 seems to be a more important target than theN domain for the process of tumor cell invasion.

Effect of MN-3, MN-15, and MN-14 Fab′ on Survival of Mice Bearing GW-39Intrapulmonary Colonic Micrometastases

GW-39 expresses both CEACAM5 and CEACAM6, as shown byimmunohistochemistry (FIG. 7, top). Based on the in vitro resultssuggesting that anti-CEACAM6 antibodies had limited antiproliferativeeffects yet showed significant anti-invasive and anti-adhesiveproperties, we pretreated GW-39 tumor cell suspensions with each of theantibody fragments (10 μg/mL) for 15 minutes before i.v. injection of304 of cells and then dosed with 100 μg of antibody on the day aftertransplantation. This design simulates the effect of a continuousexposure to antibody that would be available at any time that a cellfrom a primary tumor might initiate the metastatic cascade. The resultspresented in FIG. 7 illustrate that both MN-15 and MN-3 Fabs increasedmedian survival time (>77 and 77 days, respectively; P<0.001 versusuntreated cells) of these mice, whereas hMN-14 Fab′ (49 days) did notaffect survival significantly (42 days for untreated mice). Although thestudy was continued until mice were near death, rather than sacrificingthem at a defined time after treatment and counting the number of lungnodules, median survival time should correlate with amount of lungmetastatic disease. The results with hMN-14 Fab′ in this study aresimilar to what we reported for hMN-14 F(ab)₂ in this metastatic model(Blumenthal et al., Cancer Immuno Immunother 2005, 54:315-27),indicating that targeting the A3B3 domain does not affect mediansurvival in this model, whereas targeting the N and A1B1 domains sharedby CEACAM5 and CEACAM6 did affect metastasis and host survival when therespective Fabs are given.

Discussion

A long-term goal of immunotherapy has been to induce antitumor responsesagainst tumor-related antigens. Antibodies that directly perturbsignaling mechanisms have shown clinical benefit, such as those directedagainst the extracellular domains of HER-2/neu, epidermal growth factorreceptor, and CD20 (Weiner & Adams, Oncogene 2000, 19:6144-51; Alas etal., Cancer Res 2001, 61:5137-44). The studies presented herein suggestthat instead of being directly therapeutic by immune effector or directcytostatic mechanisms, MAbs against CEACAM6 inhibit migration, invasion,and adhesion, thereby limiting metastasis. The monovalent Fab′ form wasused for most of our studies to avoid effector cell function from the Fcregion of the MAb and focus on mechanisms implicated in the metastaticprocess.

CEACAM5 is overexpressed in a majority of carcinomas, including those ofthe gastrointestinal, respiratory and genitourinary systems, and thebreast. Our results show that another CEA family member, CEACAM6, may beas good, if not better, as a target for solid tumor antimetastatictherapy. We have shown that CEACAM6 is expressed in a larger percentageof solid tumor cell lines than CEACAM5, and the high MCF on many ofthese lines suggests that more anti-CEACAM6 MAb can be delivered tothese tumor cells. Thus, the MN-15 and MN-3 MAbs are useful for tumorsthat express CEACAM6 or CEACAM5, because they target epitopes that areshared by both antigens. These MAbs may therefore have advantages overMAbs like MN-14, which only target CEACAM5, or MAbs that are specificfor only CEACAM6 and do not cross-react with CEACAM5. Our data areconsistent with previous reports that showed a relatively high level ofCEACAM6 in the sera of a large number of lung, liver, pancreatic,breast, and colorectal cancer patients (Kuroki et al., Anticancer Res1999, 19:5599-606). Interestingly, some patients were CEACAM5−/CEACAM6+,further suggesting that CEACAM6 might be a useful independent target.

It is known that CEACAM6 is expressed on the surface of neutrophils,thus modulating adherence to endothelial-leukocyte adhesion molecule-1on activated endothelial cells (Kuijpers et al., J Cell Biol 1992,118:457-66). We have shown that MAbs to different epitopes on CEACAM6 (Nand A1B1 domains) affect cell adhesion with endothelial cells. There isalso evidence that CEACAM5 can affect cell adhesion to endothelial cells(Gangopadhyay et al. Clin Exp Metastasis 1998, 16:703-12) via activationof Kupffer cells and stimulation of IL-1β, tumor necrosis factor-α, andIL-6 production. These cytokines then induce the expression ofintercellular adhesion molecules on endothelial cells, thus increasingadhesion of tumor cells to endothelium. Our results have shown that MN-3and MN-15 Fab′ s are more active than MN-14 Fab′ at reducing adhesion ofCEACAM5+/CEACAM6+ tumor cells to endothelial cells. Thus, both the N andthe A1B1 domains but not the A3B3 domain are involved in tumor cell toendothelial cell adhesion.

Tumor cells can adhere to other tumor cells, to endothelial cells, aswell as to ECM proteins. We have found that the amount of adhesion to apanel of these proteins varied between cell lines and was not related tothe type of tumor (e.g., breast and pancreatic) or to the amount ofCEACAM5 or CEACAM6 expressed. Tumor cell interactions with ECM proteinsare important for migration and invasion and therefore metastasis. Forexample, tumor cell interactions with fibronectin are involved in thedevelopment of secondary tumors inside the bone marrow stroma via theα5β1 integrin (Van der Velde-Zimmermann et al., Exp Cell Res 1997,230:111-20). Blocking adhesion with polypeptide fragments ofheparin-binding domains of fibronectin inhibited metastasis (Matsumotoet al., Jpn J Cancer Res 1991, 82:1130-8). Similar results have beenobtained with a peptide blocking tumor cell-laminin adhesion (Islam etal., Surgery 1993, 113:676-82). Surprisingly, only MN-15 (A1B1 domain)was able to reduce adhesion to fibronectin in three of six tumor celllines tested. The percent inhibition in fibronectin adhesion in thispanel of cell lines did not correlate with the amount of adhesion inuntreated cell samples. MAbs targeting the N or A3B3 domain did notaffect cell adhesion to fibronectin. In one report by Duxbury et al. (JBiol Chem 2004, 279:23176-82), MAb-mediated CEACAM6 cross-linkingresulted in increased ECM adhesion. However, the targeted epitope wasdifferent from the ones studied here.

Active migration of tumor cells is a prerequisite of tumor cell invasionand metastasis. Adhesion molecules that increase invasion also enhancethe migratory process (Hazan et al., J Cell Biol 2000, 148:779-90).Overexpression of CEACAM6 has been reported to promote cellularinvasiveness of pancreatic cancer (Duxbury et al., Oncogene 2004,23:465-73). Agents that inhibit metastases often affect several stepsincluding migration, adhesion, and invasion. Because our data suggestthat CEACAM6 has a role in adhesion and invasion, it is important toalso assess the ability of these MAbs to impede migration. We have shownthat in cell lines with strong migratory tendencies, MAb blocking ofCEACAM5 and/or CEACAM6, with MN-3>MN-15, decreases the number ofmigrating cells. In our in vitro assay, the process of cell invasion,which involves adhesion to ECM and migration steps, was inhibited byMN-15 Fab′>MN-3 Fab′, suggesting that the A1B1 domain of CEACAM6 is moreimportant for this step but that the N domain also plays a role.

One of the notable advantages of MN-15 or MN-3 MAb therapy, comparedwith our previously reported results with MN-14 anti-CEA IgG (Blumenthalet al., Cancer Immunol Immunother 2005, 54:315-27), is the ability totarget tumors that express either CEACAM6, CEACAM5, or both, whereasMN-14 can only be used for CEACAM5+ tumors. As shown in Table 1, manysolid tumor lines express CEACAM6 but not CEACAM5 or express moreCEACAM6 than CEACAM5. These tumor types are candidates formetastasis-directed MAb therapy with CEACAM6 MAbs.

An important consideration based on the in vivo experiments is theavailability of MAb when cells first enter the circulation. MN-15 Fab′and MN-3 Fab′ showed therapeutic efficacy if cells were exposed to MAbbefore the initiation of the metastatic process. However, if MAb wasdelivered after cancer cells had exited the vasculature and had begun toseed in the lung, MAbs alone were not therapeutic (data not shown).Therefore, in certain preferred embodiments, anti-CEACAM5/CEACAM6 MAbswould be available continuously, perhaps using implantable pumps, tomaintain a desired level in the circulation.

Overall, the anti-metastasis and MAb inhibition of adhesion, invasion,and migration is a technology that should be relatively nontoxic, notlimited by issues of drug resistance, and easy to apply as an adjuvantwith other standard and/or experimental therapy approaches. BecauseCEACAM6 is also expressed in normal lung, spleen, and granulocytes(Grunert et al., Int J Cancer 1995, 63:349-55), the effect ofanti-CEACAM6 MAb on normal tissues is one consideration for therapeuticuse. In one report, CEACAM6-targeted immunotoxin therapy was effectivein a tumor-bearing nude mouse model (Duxbury et al., Biochem Biophys ResCommun 2004, 317:837-43), but this model does not express CEACAM6 onnormal tissues.

In summary, we have shown that anti-CEACAM6/CEACAM5 MAb fragments devoidof effector cell functions and targeting the N and A1B1 domains of theseantigens block migration, adhesion to endothelial cells and ECM, andinvasion, and also increase the median survival of mice withintrapulmonary micrometastases of human colonic cancer. These resultsindicate that antibodies against CEACAM5 and CEACAM6, such as MN-15, maybe efficacious in human cancer therapy.

Example 4 Expression Patterns of CEACAM5 and CEACAM6 in Primary andMetastatic Cancers

Summary

Many breast, pancreatic, colonic and non-small-cell lung carcinoma linesexpress CEACAM6 (NCA-90) and CEACAM5 (carcinoembryonic antigen, CEA),and antibodies to both can affect tumor cell growth in vitro and invivo. Here, we compare both antigens as a function of histologicalphenotype in breast, pancreatic, lung, ovarian, and prostatic cancers,including patient-matched normal, primary tumor, and metastatic breastand colonic cancer specimens.

Antigen expression was determined by immunohistochemistry (1HC) usingtissue microarrays with MN-15 and MN-3 antibodies targeting the A1B1-and N-domains of CEACAM6, respectively, and the MN-14 antibody targetingthe A3B3 domain of CEACAM5. IHC was performed usingavidin-biotin-diaminobenzide staining. The average score±SD(0=negative/8=highest) for each histotype was recorded.

For all tumors, the amount of CEACAM6 expressed was greater than that ofCEACAM5, and reflected tumor histotype. In breast tumors, CEACAM6 washighest in papillary>infiltrating ductal>lobular>phyllodes; inpancreatic tumors,moderately-differentiated>well-differentiated>poorly-differentiatedtumors; mucinous ovarian adenocarcinomas had almost 3-fold more CEACAM6than serous ovarian adenocarcinomas; lung adenocarcinomas>squamoustumors; and liver metastases of colonic carcinoma>primary tumors=lymphnodes metastases>normal intestine. However, CEACAM6 expression wassimilar in prostate cancer and normal tissues. The amount of CEACAM6 inmetastatic colon tumors found in liver was higher than in many primarycolon tumors. In contrast, CEACAM6 immunostaining of lymph nodemetastases from breast, colon, or lung tumors was similar to the primarytumor.

CEACAM6 expression is elevated in many solid tumors, but variable as afunction of histotype. Based on previous work demonstrating a role forCEACAM6 in tumor cell migration, invasion and adhesion, and formation ofdistant metastases (Blumenthal et al., Cancer Res 65: 8809-8817, 2005),it may be an important target for antibody-based therapy.

Background

The human carcinoembryonic antigen (CEA) family has 7 genes belonging tothe CEACAM subgroup. These subgroup members are mainly associated withthe cell membrane and show a complex expression pattern in normal andcancerous tissues. The CEACAM5 gene, also known as CD66e, codes for theprotein, CEA (Beauchemin et al., Exp Cell Res. 1999, 252:243-249).CEACAM5 was first described in 1965 as a gastrointestinal oncofetalantigen (Gold & Freedman, J Exp Med. 1965, 122:467-481), but is nowknown to be overexpressed in a majority of carcinomas, including thoseof the gastrointestinal tract, the respiratory and genitourinarysystems, and breast cancer (Goldenberg et al., J Natl Cancer Inst. 1976,57:11-22; Shively et al., Crit. Rev Oncol Hematol. 1985, 2:355-399;Hammarstrom, Semin Cancer Biol. 1999, 9:67-81). CEACAM6 (also calledCD66c or NCA-90) is a non-specific cross-reacting glycoprotein antigenthat shares some antigenic determinants with CEACAM5 (Kuroki et al.,Biochem Biophys Res Comm. 1992, 182:501-506). CEACAM6 also is expressedon granulocytes and epithelia from various organs, and has a broaderexpression zone in proliferating cells of hyperplastic colonic polypsand adenomas, compared with normal mucosa (Scholzel et al., Am J Pathol.2000, 157:1051-1052), as well as by many human cancers (Kuroki et al.,Anticancer Res. 1999, 19:5599-5606; Hinoda et al. J Gastroenterol. 1997,32:200-205). Relatively high serum levels of CEACAM6 are found inpatients with lung, pancreatic, breast, colorectal, and hepatocellularcarcinomas. The amount of CEACAM6 does not correlate with the amount ofCEACAM5 expressed (Kuroki et al., Anticancer Res. 1999, 19:5599-5606).

Expression of CEACAM6 in colorectal cancer correlates inversely withcellular differentiation (Ilantzis et al., Neoplasia. 2002, 4:151-163)and is an independent prognostic factor associated with a higher risk ofrelapse (Jantscheff et al., J Clin Oncol. 2003, 21:3638-3646). BothCEACAM5 and CEACAM6 have a role in cell adhesion, invasion andmetastasis, as discussed in Example 3. The present study used tissuemicroarray analysis to compare the relative expression of CEACAM5 andCEACAM6 in different histotypes of solid tumors, and compared expressionbetween primary sites and matched metastases in the same patients.

Methods

Antibodies

As discussed above, MN-15 binds to the A1B1-domain and MN-3 binds to theN-domain found on both CEACAM5 and CEACAM6. MN-14 binds to the A3B3domain only found on CEACAM5. These antibodies have similar affinitiesfor their target antigens (Hansen et al., Cancer. 1993, 71:3478-3485).MN-3 and MN-15 were used as murine MAbs, while MN-14 was included in itshumanized form, hMN-14 (labetuzumab).

Tissue Microarrays

ACCUMAX™ tissue arrays were from ISU ABXIS® (Seoul, Korea). Thefollowing arrays were used: Breast A202 (II), colon with matching livermetastases A203 (II), lung A206, pancreatic A207, prostate A208, andovary A213. Additional breast BR1001, colorectal C0991, and lung LC810arrays of matching primary tumor and lymph node metastases werepurchased from US Biomax, Inc. (Rockville, Md.). All arrays consisted ofduplicate cancer tissue cores of varying histotypes and fournon-neoplastic corresponding samples on each slide. There were 45breast, 40 lung, 26 pancreatic, 40 prostate, and 45 ovarian cancerspecimens. Some histotypes are well represented (e.g., 30 infiltratingductal breast tumors), while others had only 3-6 cores per histotype.The metastasis arrays consisted of the following matched cases: 18normal colon, primary colon cancer and liver metastases, 38 breast andlymph node metastases, 33 colon and lymph node metastases, and 37 lungand lymph node metastases.

Immunohistochemistry

Slides were deparaffinized in xylene, rehydrated, and treated with fresh0.3% hydrogen peroxide in methanol for 15 min. Following a wash in 1×phosphate-buffered saline (PBS, pH 7.4), slides were blocked with normalserum in a humid chamber for 20 min at room temperature (RT). Excessserum was rinsed off with 1×PBS and slides were incubated in a humidchamber with 25-50 μl of primary antibody (10 μg/ml) for 45 min at RT.For CEACAM5 staining, the primary antibody was murine mMN-14 IgG. ForCEACAM6 staining, slides were first blocked with humanized hMN-14 IgGand then incubated with primary antibody, either murine mMN-15 or mMN-3IgG. Excess primary antibody was washed off and sections were coveredwith biotinylated goat-anti-mouse for 30 min in a humid chamber at RT.Slides were then flooded with 0.3% H₂O₂ in methanol and 25 μlavidin-horseradish peroxidase (HRP) conjugate was added. Slides wereincubated for 45 min at RT, washed in 1×PBS, and covered with 100 μl3,3′-diaminobenzidine tetrahydrochloride solution (100 mg/mldiaminobenzide in 0.1 M sodium acetate buffer, pH 6.0, with 0.01% (v/v)H₂O₂) for 15 min. Slides were washed twice by dipping in tap water andcounterstained with 4 quick dips in hematoxylin (filtered throughWHATMAN® #4 filter paper). Slides were rinsed, air-dried, and mountedwith 1-2 drops of cytoseal and a glass coverslip. The method of Kawaiwas used to calculate a semi-quantitative score from 0 to 8 for stainingof each tissue core (Kawai et al., Clin Cancer Res. 2005, 11:5084-5089).The number of positive cells/file was estimated and assigned a number:0=none, 1= 1/100 cells, 2= 1/100 to 1/10 cells, 3= 1/10 to ⅓ cells, 4=⅓to ⅔ cells, and 5=>⅔ cells. The intensity of staining was thendetermined where 0=none, 1=weak, 2=intermediate, and 3=strong. The firstand second scores were then added together resulting in a maximumstaining score of 8 for any tissue core. Two independent blindedinvestigators performed IHC analysis and results were stronglyconsistent between the two readings. Results were recorded as themean±standard deviation for each group. Comparisons between CEACM5 andCEACAM6 scores for a given histotype or between histotypes for eachantigen were assessed by a one-factor analysis of variance with the useof a two-tailed F test and a 95% confidence limit. The null hypothesisHo: μ1=μ2=¼ μk, where k equals the number of experimental groups, wasused. A two-tailed test takes into account an extreme value in any onegroup that deviates from the population mean in either the high or lowdirection (two-sided). The F value is a measure of the probability thatthis difference in groups could occur by chance alone.

Results

Expression in Solid Tumors as a Function of Histotype

For all tumor cores evaluated, the amount of CEACAM6 was greater thanthat of CEACAM5. However, the homogeneity of expression and stainingintensity varied between tissue histotypes and between samples withinthe same histological type. We evaluated 45 breast tumor cores: 30infiltrating ductal carcinoma, 8 papillary, 4 lobular, and 3 phyllodes.CEACAM6 levels were higher than CEACAM5 levels for all histotypes(P<0.001). The highest CEACAM6 expression was found in papillary(6.0±2.1)>infiltrating ductal (5.1±2.5)>lobular (4.0±0.8)>phyllodes(2.0±1.0). The differences between papillary and lobular breast cancerswere significant at the P<0.01 level. The highest CEACAM5 expression wasfound in papillary samples (1.4±1.4), but was not statisticallydifferent from infiltrating ductal or lobular samples. Pyllodes breastcancer is a stromal tumor, usually benign, and should therefore notexpress CEACAM5 or CEACAM6.

CEACAM5 and CEACAM6 expression was assessed in 6 different lung cancers:5 each of well, moderately and poorly differentiated adenocarcinoma, 5each of well, moderately and poorly differentiated squamous carcinoma, 3each of large cell and bronchioalveolar, and 2 each of large cellneuroendocrine and small cell cancer. Among these, adenocarcinomaexpressed more CEACAM6 than squamous cancer (P<0.001). The highestCEACAM6 expression was found in moderately-differentiated adenocarcinoma(7.8±0.4)>well-differentiated adenocarcinoma (7.3±1.1)=bronchioalveolar(7.2±0.8)>poorly-differentiated adenocarcinoma (6.8±1.0)>small-cell(5.5±0.7)>well-differentiated squamous(5.2±1.0)>moderately-differentiated squamous cancer (4.9±1.1). CEACAM6levels in large-cell (4.5±0.9) and poorly-differentiated squamouscarcinomas (3.8±1.3) were similar to non-neoplastic lung tissue (P═NS),suggesting that anti-CEACAM6 antibodies would not be effective withthese histotypes of lung cancer. The highest expression of CEACAM5 wasin small-cell lung cancer specimens (5.5±0.7), followed by large-cellneuroendocrine tumors (4.75±3.18). Large-cell tumors were CEA-negativeand all adenocarcinomas and serous tumors scored ≦2.60.

Pancreatic cancer has been the most extensively studied neoplasm withrespect to CEACAM6 expression. We evaluated CEACAM5 and CEACAM6 inpancreatic cancer as a function of tumor cell differentiation. Onewell-differentiated, 3 well-moderately differentiated, 13moderately-differentiated, 2 moderately- to poorly-differentiated, and 7poorly-differentiated tumor cores were studied. The highest expressionof CEACAM6 in pancreatic tumors was found inmoderately-(7.5±0.7)>moderately-poor (5.9±1.9)=well-moderatelydifferentiated (5.8±1.8)>poorly-differentiated tumors(5.1±2.5)>well-differentiated (4.0±0.0) adenocarcinomas (P═NS betweenthe subtypes). Non-neoplastic pancreas CEACAM6 expression was 2.25±0.5.The well-moderately, moderately, and moderately-poor adenocarcinomaswere significantly higher than non-neoplastic pancreas (P<0.001).CEACAM6 expression did not correlate with disease stage. Samples withhigh (8) and low (3-4) expression could be found in stages IA-IB,IIA-IIB, and IV. CEACAM5 expression was lower than CEACAM6 for allhistotypes; the highest expression being found in moderatelydifferentiated tumors (4.0±1.4) and the least in the moderate-poor(0.92±1.92) and poorly-differentiated (1.4±1.5) tumors. Only themoderately and the well-moderately differentiated tumors expressedsignificantly more CEACAM5 than non-neoplastic tissues (P<0.002 andP<0.005, respectively).

Eighteen stage-II, 15 stage-III, and 4 stage-IV prostate tumor coreswere stained for CEACAM5 and CEACAM6. Gleason scores of 4 to 9 wererepresented in the stage-II samples, and Gleason scores of 6 to 10 werefound in the stage-III specimens. All stage-IV samples were Gleason9-10. Expression did not correlate with Gleason score of the samplewithin any stage. Similar expression of CEACAM6 was found in stage-II,-III, and -IV prostate cancer (3.3-3.8), and was not significantlydifferent from non-neoplastic prostate tissue (P═NS). CEACAM5 expressionwas consistently below 0.9 for all stages of prostate cancer and was notgreater than expression levels in non-neoplastic prostate tissue(0.5±1.0; P═NS).

Nine ovarian cancer types were studied: 5 each of serous adenocarcinoma,mucinous adenocarcinoma, clear cell carcinoma, and transitional cellcarcinoma; 4 each of endometrioid adenocarcinoma and Brenner tumor; and3 each of yolk sac tumor, granulosa cell tumor, and dysgerminoma. Theamount of CEACAM6 in ovarian cancer was highest in mucinousadenocarcinoma (5.6±1.7)>transitional cell (3.8±1.6)>endometrioid(3.6±2.8)>clear cell (3.4±1.8)>yolk sac tumors (2.5±0.5). Mucinous tumorCEACAM6 expression was significantly higher than transitional andendometroid (P<0.02), clear cell (P<0.01), and yolk sac tumors(P<0.005). Much lower levels were found in serous adenocarcinoma(1.8±1.3)>Brenner tumor (1.3±1.4)=dysgerminoma (1.3±1.5)>granulosa cell(0.7±1.2). Normal ovary samples were negative for CEACAM6. Thus, alltumor histotypes expressed significantly more CEACAM6 thannon-neoplastic ovary. The highest CEACAM5 expression also was found inthe mucinous adenocarcinoma type (1.6±1.5). Expression of CEACAM5 in allother ovarian samples scored below 0.6, and non-neoplastic ovary scoreswere 0.5±1.0. Mucinous CEACAM5 levels were significantly higher than allother histotypes (P<0.002 compared with endometroid and Brenner tumors,and P<0.001 compared with serous, clear cell, transitional and yolksac).

Much larger amounts of CEACAM6 were found in colon adenocarcinoma(6.2±1.4) compared with non-neoplastic colon (3.0±0.0; P<0.002) andCEACAM6 expression exceeded CEACAM5 expression (3.4±0.5; P<0.001).

CEACAM6 expression has been associated with cell adhesion, a key step inthe metastatic cascade. We have shown above that antibody to CEACAM6expression can block adhesion. Therefore, we assessed whether CEACAM6expression was similar or different between matched primary colon andmetastatic liver sites. In half of the matched cases (N=6), CEACAM6expression was much greater in the liver metastasis than in the primarycolon tumors, and in the remaining 6 cases, the amounts were comparablebetween the primary and the metastatic liver sites.

In contrast to the higher expression of CEACAM6 in many secondary liversites from colon cancer, there was no pattern for CEACAM6 expressionbetween primary tumor and lymph node metastases. For breast samples, thelymph node sites had higher CEACAM6 expression in 7 pairs, lower CEACAM6in 6, and no difference in 25 pairs. For lung samples, the lymph nodesites had higher CEACAM6 expression in 10 pairs, lower CEACAM6 in 11,and no difference in 16 pairs. For colon samples, the lymph node siteshad higher CEACAM6 expression in 7 pairs, lower CEACAM6 in 10, and nodifference in 11 pairs.

Discussion

CEACAM5 and CEACAM6 are two tumor-associated antigens that playimportant regulatory roles in cell adhesion and in tumor cellchemosensitivity (Glinsky, Cancer Metastasis Rev. 1998, 17:177-186;Kraus et al., Oncogene. 2002, 21:8683-8695; Zhou et al., Cancer Res.1993, 53:3817-3822). CEACAM6 overexpression independently predicts pooroverall survival and poor disease-free survival, whereas CEACAM5 has notbeen related significantly to these outcomes (Jantscheff et al., Vol.37. Eur J Cancer, 2001, 37:S290).

Studies have shown that CEACAM5 affects expression of various groups ofcancer-related genes, especially cell cycle and apoptotic genes,protecting colonic tumor cells from various apoptotic stimuli, such astreatment with 5-fluorouracil (Soeth et al., Clin Cancer Res. 2001,7:2022-2030). Therefore, CEACAM5 expression may be a means for cancercells to overcome apoptosis-inducing therapies. Ordonez et al. havereported that expression of both CEACAM5 and CEACAM6 plays a role ininhibiting apoptosis of cells when deprived of their anchorage to theextracellular matrix, a process known as anoikis (Ordonez et al., CancerRes. 2000, 60:3419-3424). Increased expression of CEACAM6 correlateswith a decrease in sensitivity to drugs, like gemcitabine (Duxbury etal., Cancer Res. 2004, 64:3987-3993). Targeting CEACAM5 and/or CEACAM6may therefore be a novel method of modulating cancer cellchemosensitivity and apoptosis. It has been reported that siRNA toCEACAM6 impairs resistance to anoikis and increases caspase-mediatedapoptosis of xenografted tumors (Duxbury et al., Oncogene. 2004,23:465-473). Antibody-directed targeting of CEACAM6 may provide aclinically feasible alternative to RNA interference silencing to enhanceresponsiveness to chemotherapeutic agents in those tumors that expressCEACAM6.

To determine which solid tumors and histological types would be mostamenable to antibody blocking of CEACAM5 and CEACAM6, we studiedexpression of these antigens using tissue microarray analysis. To date,pancreatic and colonic cancer have been the focus of CEACAM6 expressionin the literature (Duxbury et al., Ann Surg. 2005, 241:491-496; Koderaet al., Br J Cancer. 1993, 68:130-136). Here, we have further exploredthe expression of CEACAM6 in a panel of solid tumors: breast, lung,ovary and prostate cancer, in addition to expanding on pancreatic andcolonic tumors, and used tissue microarrays to further define tumorsthat are CEACAM6+ as a function of histological type in all six solidtumor categories. Our results show that expression is strongly dependenton the histotype of the tumor. Antigen expression in some subtypes is2-4-fold higher than in normal tissues, while in others, expression issimilar to non-neoplastic tissues.

The demonstration of higher CEACAM6 expression compared with CEACAM5across most solid tumors, and the differential expression as a functionof histotype, are important observations for translating anti-CEACAM6therapy to patients. This analysis is a step towards elucidating theimportance of CEACAM6 as a tumor target in a variety of solid tumorsthat extend the many important studies reported for pancreatic cancer(Duxbury et al., J Biol. Chem. 2004, 279:23176-23182; Duxbury et al.,Cancer Res. 2004, 64:3987-3993; Duxbury et al., Biochem Biophys ResComm. 2004, 317:837-843; Duxbury et al., Ann Surg. 2005, 241:491-496).It also reveals that expression level varies as a function of tumorhistotype.

We have also addressed the expression pattern of CEACAM6 in primarytumors and in matched metastases in the same patients. Our results showthat in half of the clinical specimens, liver metastases had a muchhigher expression of CEACAM6 than the primary colorectal tumors,suggesting that in such patients, blocking adhesion and invasion thatresults from CEACAM6 expression might influence the ability of tumorcells to metastasize, as we have in fact shown experimentally(Goldenberg et al., J Natl Cancer Inst. 1976, 57:11-22). However,CEACAM6 expression in lymph node metastases was similar to the amount ofantigen in primary breast, colon or lung tumor samples. The mechanism bywhich malignant tumors invade lymphatics and metastasize to regionallymph nodes appears to be regulated by VEGF-C and VEGF-D inducedlymphogenesis (Detmar & Hirakawa, J Exp Med. 2002, 196:713-718) and achemokine gradient. Directional movement is related to chemokinereceptor expression on tumor cells (Nathanson, Cancer. 2003,98:413-423), but does not involve members of the CEACAM family. Incontrast, CEACAM6 plays an important role in migration, invasion andadhesion (Duxbury et al., Oncogene. 2004, 23:465-473), steps that areimportant in the metastatic spread to secondary tissue sites other thanlymph nodes. Anti-adhesive molecules that disrupt cell-matrix andcell-cell attachments have been proposed as potential cancertherapeutics based on their ability to interfere with motility,adhesion, and metastatic progression (Blumenthal et al., Cancer Res.2005, 65:8809-8817; Glinsky, Cancer Metastasis Rev. 1998, 17:177-186;Kerbel et al., Bulletin de I'institut Pasteur. 1995, 92:248-256).

We have reported that the humanized anti-CEA (CEACAM5) antibody, hMN-14,can enhance the therapeutic effects of two cytotoxic drugs usedfrequently in colorectal cancer therapy, fluorouracil and CPT-11, inboth subcutaneous and metastatic human colonic tumor cells propagated innude mice (Blumenthal et al., Cancer Immuno Immunother. 2004,54:315-27). In another high CEA-expressing human medullary thyroidcancer xenograft, we have also shown that MN-14 anti-CEA IgG can inhibittumor cell growth and also augment the effects of dacarbazine, a drugthat is active in this cancer type (Stein et al., Mol Cancer Ther. 2004,2:1559-1564). One explanation may involve a role in antibody blockingadhesion (Zhou et al., Cancer Res. 1993, 53:3817-3822) and therebychemosensitizing the tumor cells.

In a series of studies, Duxbury and associates have shown that silencingCEACAM6 by siRNA: (a) enhances cell anoikis, (b) increases caspaseactivation in response to anchorage independent conditions, (c)downregulates the Akt cell survival pathway, (d) inhibits metastasis invivo, and (e) enhances gemcitabine induced chemosensitivity (Duxbury etal., Cancer Res. 2004, 64:3987-3993; Duxbury et al., Oncogene. 2004,23:465-473; Duxbury et al., Biochem Biophys Res Comm. 2004, 317:133-141;Duxbury et al., Ann Surg. 2005, 241:491-496). Thus, in addition toCEACAM5, CEACAM6 may also be a useful therapeutic target. BlockingCEACAM6-mediated homotypic and/or heterotypic adhesion may haveanti-metastatic and chemosensitizing effects. In ongoing preclinicaltherapy studies, we are examining the therapeutic effects ofunconjugated anti-CEACAM6 antibody alone or combined with standardchemotherapeutic agents in colon, breast, and lung metastasis models. Analternative approach is to develop an anti-CEACAM6 immunoconjugate as atherapeutic agent for CEACAM6+ tumors (Duxbury et al. Biochem BiophysRes Comm. 2004, 317:837-843). In vitro targeting with an anti-CEACAM6antibody, followed by secondary saporin-conjugated immunoglobulin (IgG),induced marked cytotoxicity via caspase-mediated apoptosis. In an invivo nude mouse xenograft model, this indirect immunotoxin approachmarkedly suppressed pancreatic adenocarcinoma tumor growth and enhancedtumor apoptosis.

Conclusion

Based on expression level, CEACAM6 may be a more promising target forantibody-based anti-metastatic and chemosensitizing therapy than CEACAM5in the solid tumors studied. Furthermore, CEACAM6 may be a usefulantigen to target in select subtypes of solid tumors. In colonic cancer,CEACAM6 may play an important role in the development of distantmetastases.

Example 5 In Vivo Effect of Pretreatment with an Immunomodulator Priorto Treatment With hMN-15 and CPT-11 on Tumor Cell Chemosensitivity

The effect of combined treatment of hMN-15 with CPT-11 is evaluated,initiated together in mice with GW-39 tumors expressing higher CEAlevels, as a result of pretreatment of GW-39 stock tumors (10% GW-39cell suspension) with interferon-γ (IFNγ). The experiments involvinginterferon-gamma enhancing the antitumor effects of naked CEA antibody(hMN-15) are conducted as follows.

GW-39 human colon cancer is grown subcutaneously in a mouse thatreceives 100,000 units of IFN-gamma twice a day for 4 days. A controlmouse with GW-39 tumor is not given IFN. Experimental mice are injectedi.v. with a 5% suspension of GW-39 (w/v) from either of the two mice(i.e., with or without IFN treatment) into two groups of eight. Four ofeach receive tumor from the IFN-treated mice and four from the untreatedmice. One group of 8 mice then receive hMN-15 (100 μg per day×14 daysand then twice weekly thereafter until experiment is ended), anothergroup receive CPT-11 at 160 μg/day×5 days (=20% of maximum tolerateddose), a third group receives the same doses of antibody+drug combined,and a fourth group is not treated at all. Animal weights are measuredand survival determined weekly. Also, samples of stock tumor treatedwith IFN in the mice that are later implanted are also processed forimmunohistology to assess increase in CEA expression in the tumors frommice treated with IFN-gamma, and this is controlled by also treating thesuspensions by immunohistology with an irrelvant IgG, such as Ag8, whichshows no CEA staining.

It is observed that IFNγ pretreatment increases the expression of CEA inGW-39 tumors. The combination of naked hMN-15 with CPT-11 is moreeffective at reducing tumor growth and increasing survival than the sumof the effects of either therapeutic agent alone. This effect is morepronounced in the tumors pretreated with IFNγ.

Example 6 Sigmoid Colon Cancer Therapy with Anti-CEA Antibody and GM-CSF

JR is a 62-year-old man who is refractive to chemotherapy with5-fluorouracil and leucovorin to reduce his metastases to the liverfound at the time of removal of his sigmoid colon cancer. His plasmatiter of carcinoembryonic antigen (CEA) at presentation is 34 ng/mL, andcomputed tomography of the liver shows several small lesions measuringbetween 2 and 4 cm in diameter in the right lobe. Other radiologicalstudies appear to be normal. Immunotherapy with humanized anti-CEA IgG(hMN-15) monoclonal antibody is begun on a weekly basis for 4 weeks, atan intravenous dose of 300 mg/m² infused over 2 hours. One week prior tohMN-15 therapy, the patient receives 2 subcutaneous injections of 200μg/m² GM-CSF, 3 days apart, and continued twice weekly during the 4weeks of hMN-15 therapy. After these four weeks, both hMN-15 and GM-CSFare given at the same doses every other week for an additional 3 months,but the dose of GM-CSF is increased to 250 μg/m². Prior to eachadministration of the humanized anti-CEA antibody, the patient is givendiphenhydramine (50 mg orally), and acetaminophen (500 mg orally).

At this time, the patient is restaged, with CT measurements made of theliver metastases and diverse radiological scans of the rest of the body.Blood is also taken for chemistries and for determination of his bloodCEA titer. No areas of disease outside of the liver are noted, but thesum of the diameters of the measurable tumors in the liver appear todecrease by 40 percent, and the patient's blood CEA titer decreases to18 ng/mL, thus indicating a therapeutic response.

Immunotherapy with hMN-15 and GM-CSF, given once every other week at 200mg/m² for hMN-15 and 250 μg/m² for GM-CSF, are administered for another2 months, and restaging shows additional decrease in the sum of thediameters of the liver tumors and a fall in the CEA titer to 10 ng/mL.Since tumor decrease is measured as being >65% over the pre-therapybaseline, the therapy is considered to have provided a partial response.After this, the doses are made less frequent, once every month for thenext six months, and all studies indicate no change in disease. Thepatient is then followed for another 10 months, and remains in a partialremission, with no adverse reactions to the therapy, and generallywithout any symptoms of disease.

Example 7 Combined Immunotherapy and Chemotherapy of Metastatic ColonCancer

ST is a 52-year-old woman presenting with liver and lung metastases ofcolon cancer following resection of the primary tumor. She is placed ona combined chemotherapy and immunotherapy protocol based on the Gramontschedule (de Gramont et al., J Clin Oncol. 2000, 18:2938-47), but withthe addition of humanized anti-CEA monoclonal antibody IgG₁ (hMN-15).Prior to infusions of the antibody, she receives 50 mg orally ofdiphenhydramine and 500 mg orally of acetaminophen. She receives a 2-hrinfusion of leucovorin (200 mg/m²/day) followed by a bolus of5-fluorouracil (400 mg/m²/day) and 22-hour continuous infusion of5-fluorouracil (600 mg/m²/day) for 2 consecutive days every 2 weeks,together with oxaliplatin at 85 mg/m² as a 2-hr infusion in 250 mL ofdextrose 5%, concurrent with leukovorin on day 1 (FOLFOX4 schedule). Thepatient also receives anti-emetic prophylaxis with a5-hydroxyltryptamine-3-receptor antagonist. One week prior to this2-week chemotherapy cycle, hMN-15 monoclonal anti-CEA antibody isinfused over 2 hrs at a dose of 200 mg/m², and repeated each week of the2-week chemotherapy cycle, and every week thereafter for the next monthwith another chemotherapy cycle. Also, a subcutaneous dose of 5μg/kg/day of G-CSF is administered once weekly beginning with the secondchemotherapy cycle, and continued at this dose for the duration ofimmunotherapy with hMN-15 antibody, over the next 3 months.

A total of 5 cycles of chemotherapy is performed with continuedadministration of hMN-15 antibody and filgrastim. Thereafter, hMN-15 andfilgrastim therapy is given, at the same doses, every other week for thenext 3 months, without chemotherapy. The patient is staged 2 monthslater, and her liver and lung metastases show shrinkage by computedtomography measurements of >80 percent of disease measured in the liverand lungs, as compared to the measurements made prior to therapy. Herblood CEA titer also shows a drop from the pre-therapy level of 63 ng/mLto 9 ng/mL. She is followed over the next 6 months, and her diseaseappears to be stable, with no new lesions found and no increase in thedisease remaining in the liver and lungs. The patient's predominanttoxicity is peripheral sensory neuropathy, which consists oflaryngeopharyngeal dysesthesia. The patient also experiences diarrhea,mucositis, nausea and vomiting during the chemotherapy cycles, but theseare not excessive. She does not experience any adverse events when onlyimmunotherapy is administered, and is able to return to full-timeactivities without any significant restrictions.

Example 8 Effect of hMN-15 Pretreatment on CPT-11 Efficacy

The effects of pre-treatment with naked hMN-15 CEA Mab given 3 daysprior to CPT-11 treatment is examined in a SUM1315 breast cancer model(Kuperwasser et al., Cancer Res. 2005, 65:6130-38). CPT-11 alone, hMN-15alone, and combination therapy of hMN-15+CPT-11 where the hMN-15 isadministered 3 days prior to the CPT-11 are compared. Dosages are asindicated in Example 5. hMN-15 alone increases median survival time by21% under these conditions. CPT 11 alone increases survival by 76%. Bycontrast, the combination therapy where hMN-15 is administered 3 daysprior to CPT-11 produces a median survival time increase of anadditional 58% above CPT-11 alone. Pre-treatment with hMN-15significantly prolongs survival of animals with low tumor burden in ametastatic model of human breast cancer. The synergistic effect ofhMN-15 antibody with CPT-11 therapy is surprising.

Example 9 Effect of Administration Schedule on hMN-15 Synergy withCPT-11

A comparison is made of various administration schedules of naked hMN-15CEA Mab and CPT-11 in a human colon cancer model. Giving hMN-15 3 daysbefore CPT-11 is the most effective. Dosages are as indicated Example 5.When the order is reversed (CPT-11 is given 3 days before hMN-15) orwhen both are given together at the same time, median survival time of70 days is an increase over the untreated control group (35 days) but isstill less than the median survival time of 105 days with the hMN-15pre-treatment 3 days before CPT-11.

Example 10 Class I Anti-CEA Antibodies and Medullary Thyroid CancerTherapy

TT, a human medullary thyroid cell line, is purchased from the AmericanType Culture Collection. The cells are grown as monolayers in DMEM (LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal bovineserum, penicillin (100 u/ml), streptomycin (100 μg/ml), and L-glutamine(2 mM). The cells are routinely passaged after detachment with trypsin,0.2% EDTA.

Tumors are propagated in female nu/nu mice (Taconic Farms, Germantown,N.Y.) at 6-8 weeks of age by s.c. injection of 2×10⁸ washed TT cells,which is propagated in tissue culture. Antibodies are injected i.v., viathe lateral tail vein, into the tumor-bearing animals. Tumor size ismonitored by weekly measurements of the length, width, and depth of thetumor using a caliper. Tumor volume is calculated as the product of thethree measurements.

To study whether naked hMN-15 can add to the efficacy of DTIC, TTbearing nude mice are given DTIC (75 μg/dose) in combination with acourse of treatment of the unlabeled MAb. DTIC is administered for 3consecutive days at 75 μg/dose as one course, beginning 2 days afters.c. injection of TT cells. hMN-15 MAb treatment is initiated 3 daysprior to the first dose of DTIC, at 100 μg/dose/day for 5 days in thefirst two weeks, then twice weekly. Significant delays in tumor growthare caused by these schedules of either MAb therapy or chemotherapyalone. Surprisingly, the 75-μg dose of DTIC in combination with thisschedule of hMN-15 is more effective than either treatment alone. At 7weeks, 8/10 mice in the 75 μg DTIC+MAb group have no palpable tumor,compared to 1/10 in the 75 μg DTIC-only group and 0/10 in the untreatedand MAb-only groups. Mean tumor volumes at 7 weeks are 0.018+0.039 cm³(75 μg DTIC+hMN-15), 0.284+0.197 cm³ (75 μg DTIC), 0.899+0.545 cm³(hMN-15) and 1.578+0.959 cm³ (untreated).

Example 11 Preparation of DNL Constructs for Pretargeting

In various forms, the DNL technique may be used to make dimers, trimers,tetramers, hexamers, etc. comprising virtually any antibodies orfragments thereof or other effector moieties, such as cytokines. Forcertain preferred embodiments, IgG antibodies or Fab antibody fragmentsmay be produced as fusion proteins containing either a DDD or ADsequence. Bispecific antibodies may be formed by combining a Fab-DDDfusion protein of a first antibody with a Fab-AD fusion protein of asecond antibody. Alternatively, constructs may be made that combineIgG-AD fusion proteins with Fab-DDD fusion proteins. For purposes ofpre-targeting, an antibody or fragment containing a binding site for anantigen associated with a target tissue to be treated, such as a tumor,may be combined with a second antibody or fragment that binds a haptenon a targetable construct. In exemplary embodiments, the tumor targetingantibody or fragment is a chimeric, humanized or human MN-15 antibodycomprising the 6 MN-15 CDR sequences, while the hapten binding moietymay be an anti-HSG or anti-DTPA antibody. The bispecific antibody (DNLconstruct) is administered to a subject, circulating antibody is allowedto clear from the blood and localize to target tissue, and a targetableconstruct attached to at least one therapeutic and/or diagnostic agentis added and binds to the localized antibody.

Independent transgenic cell lines may be developed for each Fab or IgGfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD₂-fusion protein module can be combined with any AD-fusionprotein module to generate a bispecific DNL construct. For differenttypes of constructs, different AD or DDD sequences may be utilized.

DDD1: (SEQ ID NO: 11) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2:(SEQ ID NO: 12) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1:(SEQ ID NO: 13) QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 14)CGQIEYLAKQIVDNAIQQAGC

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. (See, Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.)The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD1, SEQ ID NO:11). To generate Fab-AD expression vectors, thesequences for the hinge, CH₂ and CH3 domains of IgG are replaced with asequence encoding the first 4 residues of the hinge, a 15 residueGly-Ser linker and a 17 residue synthetic AD called AKAP-IS (referred toas AD1, SEQ ID NO:13), which was generated using bioinformatics andpeptide array technology and shown to bind RIIα dimers with a very highaffinity (0.4 nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A(2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC) followed by four glycinesand a serine, with the final two codons (GS) comprising a Bam HIrestriction site. The 410 by PCR amplimer was cloned into the pGemT PCRcloning vector (Promega, Inc.) and clones were screened for inserts inthe T7 (5′) orientation.

Construction of (G₄S)₂DDD1 ((G₄S)₂ Disclosed as SEQ ID NO: 15)

A duplex oligonucleotide, designated (G₄S)₂DDD1 ((G₄S)₂ disclosed as SEQID NO: 15), was synthesized by Sigma Genosys (Haverhill, UK) to code forthe amino acid sequence of DDD1 preceded by 11 residues of the linkerpeptide, with the first two codons comprising a BamHI restriction site.A stop codon and an EagI restriction site are appended to the 3′ end.The encoded polypeptide sequence is shown below.

(SEQ ID NO: 16) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFT RLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, thatoverlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGenosys) and combined to comprise the central 154 base pairs of the 174by DDD1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase. Following primerextension, the duplex was amplified by PCR. The amplimer was cloned intopGemT and screened for inserts in the T7 (5′) orientation.

Construction of (G₄S)₂-AD1 ((G₄5)₂ Disclosed as SEQ ID NO: 15)

A duplex oligonucleotide, designated (G₄S)-2-AD1 ((G₄5)₂ disclosed asSEQ ID NO: 15), was synthesized (Sigma Genosys) to code for the aminoacid sequence of AD1 preceded by 11 residues of the linker peptide withthe first two codons comprising a BamHI restriction site. A stop codonand an EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 17) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the pGemT vector and screened for inserts in the T7 (5′)orientation.

Ligating DDD1 with CH1

A 190 by fragment encoding the DDD1 sequence was excised from pGemT withBamHI and NotI restriction enzymes and then ligated into the same sitesin CH1-pGemT to generate the shuttle vector CH1-DDD1-pGemT.

Ligating AD1 with CH1

A 110 by fragment containing the AD1 sequence was excised from pGemTwith BamHI and NotI and then ligated into the same sites in CH1-pGemT togenerate the shuttle vector CH1-AD1-pGemT.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-based vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-15-pdHL2

C-DDD1-Fd-hMN-15-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-15, in which DDD1 is linked to hMN-15 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN15-pdHL2, which is used to produce hMN-15 IgG, is converted toC-DDD1-Fd-hMN-15-pdHL2 by digestion with SacII and EagI restrictionendonucleases to remove the CH1-CH3 domains and insertion of theCH1-DDD1 fragment, which is excised from the CH1-DDD1-SV3 shuttle vectorwith SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

C-DDD2-Fd-hMN-15-pdHL2

C-DDD2-Fd-hMN-15-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-15, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-15via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-15 Fab held togetherby non-covalent interaction of the DDD2 domains.

The expression vector is engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide (GGGGSGGGCG, SEQ ID NO:18) and residues 1-13of DDD2 (SEQ ID NO:12), are made synthetically. The oligonucleotides areannealed and phosphorylated with T4 PNK, resulting in overhangs on the5′ and 3′ ends that are compatible for ligation with DNA digested withthe restriction endonucleases BamHI and PstI, respectively.

The duplex DNA is ligated with the shuttle vector CH1-DDD1-pGemT, whichis prepared by digestion with BamHI and PstI, to generate the shuttlevector CH1-DDD2-pGemT. A 507 by fragment is excised from CH1-DDD2-pGemTwith SacII and EagI and ligated with the IgG expression vectorhMN15-pdHL2, which is prepared by digestion with SacII and EagI. Thefinal expression construct is designated C-DDD2-Fd-hMN-15-pdHL2. Similartechniques have been utilized to generated DDD2-fusion proteins of theFab fragments of a number of different humanized antibodies.

H679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-15 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchor domain sequence of AD2 (SEQ IDNO:14) appended to the carboxyl terminal end of the CH1 domain via a 14amino acid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-pGemT, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-pGemT. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Generation of Trimeric MN-15-679 Construct

A trimeric DNL construct is obtained by reacting C-DDD2-Fab-hMN-15 withh679-Fab-AD2. A pilot batch of DNL trimer is generated with >90% yieldas follows. Protein L-purified C-DDD2-Fab-hMN-15 (200 mg) is mixed withh679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. The total proteinconcentration is 1.5 mg/ml in PBS containing 1 mM EDTA. Subsequent stepsinvolved TCEP reduction, HIC chromatography, DMSO oxidation, and IMP 291affinity chromatography. Addition of 5 mM TCEP rapidly results in theformation of a₂b complex consistent with a 157 kDa protein expected forthe binary structure. The trimeric DNL construct is purified to nearhomogeneity by IMP 291 affinity chromatography. IMP 291 is a syntheticpeptide containing the HSG hapten to which the 679 Fab binds (Rossi etal., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLC analysis of the IMP291 unbound fraction demonstrates the removal of a₄, a₂ and free kappachains from the product. Binding studies indicate that the trimeric DNLconstruct incorporating the hMN-15 antibody binds to CEA and HSG, withaffinities similar to the parent MN-15 and 679 antibodies.

Production of TF10 Bispecific Antibody

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The cancer-targeting antibody component in TF10 isderived from hPAM4, a humanized anti-pancreatic cancer mucin MAb thathas been studied in detail as a radiolabeled MAb (e.g., Gold et al.,Clin. Cancer Res. 13: 7380-7387, 2007). The hapten-binding component isderived from h679, a humanized anti-histaminyl-succinyl-glycine (HSG)MAb discussed above. The TF10 bispecific ([hPAM4]₂×h679) antibody wasproduced using the method disclosed for production of the (anti CEA)₂×anti HSG bsAb, as described above. The TF10 construct bears twohumanized PAM4 Fabs and one humanized 679 Fab.

The two fusion proteins (hPAM4-DDD and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD. The reaction mixture was incubated at room temperature for24 hours under mild reducing conditions using 1 mM reduced glutathione.Following reduction, the DNL reaction was completed by mild oxidationusing 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

hMN-15-Fd-AD2-pdHL2

For certain DNL constructs, it is preferred to use an hMN-15-Fab-AD2fusion protein, which possesses an anchor domain sequence of AD2 (SEQ IDNO:14) appended to the carboxyl terminal end of the CH1 domain via a 14amino acid residue Gly/Ser peptide linker. An hMN-15-Fab-AD2 constructmay be utilized, for example, to prepare a DNL construct comprising alower number of hMN-15 subunits and a greater number of other effectormoiety subunits, such as a toxin, cytokine, drug or another antibody orantibody fragment.

The expression vector is engineered as described above for theh679-Fab-AD2 fusion protein. Two overlapping, complimentaryoligonucleotides (AD2 Top and AD2 Bottom), which comprise the codingsequence for AD2 and part of the linker sequence, are madesynthetically. The oligonucleotides are annealed and phosphorylated withT4 PNK, resulting in overhangs on the 5′ and 3′ ends that are compatiblefor ligation with DNA digested with the restriction endonucleases BamHIand SpeI, respectively.

The duplex DNA is ligated into the shuttle vector CH1-AD1-pGemT, whichis prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-pGemT. A 429 base pair fragment containing CH1 and AD2coding sequences is excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into hMN-15-pdHL2 vector prepared bydigestion with those same enzymes. The final expression vector ishMN-15-Fd-AD2-pdHL2.

Example 12 Imaging Using Pretargeting with hMN-15-679 DNL Construct and¹¹¹In-Labeled Peptides

The following study demonstrates the feasibility of in vivo imagingusing the pretargeting technique with labeled targeting peptides andbispecific antibodies incorporating hMN-15. The hMN-15-679 DNLconstruct, comprising two copies of a C-DDD2-Fab-hMN-15 and one copy ofC-AD2-Fab-679, is prepared as described in the preceding Example. Nudemice bearing 0.2 to 0.3 g human colon cancer xenografts are imaged,using pretargeting with the hMN-15-679 DNL construct and an¹¹¹In-IMP-288 peptide. The results show clearly delineated tumors inanimal models using a bsMAb pretargeting method. Experimental animalsreceive different doses of hMN-15-679 DNL construct at 10:1 or 20:1 moleratio to the moles of peptide given, and the next day they are given an¹¹¹¹n-labeled diHSG peptide (IMP 288). The control animals receive onlythe ¹¹¹In-IMP-288 (no pretargeting). The images are taken 3 h after theinjection of the labeled peptide and show clear localization of 0.2-0.3g tumors in the pretargeted animals with both doses of DNL construct,with no localization in the animals given the ¹¹¹In-peptide alone.

Example 13 PEGylated DNL Constructs

In certain embodiments, it may be preferred to prepare constructscomprising PEGylated forms of antibody or immunoconjugate, for exampleto increase the serum half-life of the antibody or immunoconjugatemoiety. Such PEGylated constructs may be prepared by the DNL technique.

In a preferred method, the effector moiety to be PEGylated, such ashMN-15 Fab, is linked to a DDD sequence to generate the DDD module. APEG reagent of a selected molecular size is derivatized with acomplementary AD sequence and the resulting PEG-AD module is combinedwith the DDD module to produce a PEGylated conjugate that consists of asingle PEG tethered site-specifically to two copies of the hMN-15 Fab orother effector moiety via the disulfide bonds formed between DDD and AD.The PEG reagents may be capped at one end with a methoxy group (m-PEG),can be linear or branched, and may contain one of the followingfunctional groups: propionic aldehyde, butyric aldehyde,ortho-pyridylthioester (OPTE), N-hydroxysuccinimide (NHS),thiazolidine-2-thione, succinimidyl carbonate (SC), maleimide, orortho-pyridyldisulfide (OPPS). Among the effector moieties that may beof interest for PEGylation are enzymes, cytokines, chemokines, growthfactors, peptides, aptamers, hemoglobins, antibodies and antibodyfragments. The method is not limiting and a wide variety of agents maybe PEGylated using the disclosed methods and compositions. PEG ofvarious sizes and derivatized with a variety of reactive moieties may beobtained from commercial sources, such as NEKTAR® Therapeutics(Huntsville, Ala.).

Generation of PEG-AD2 Modules

(SEQ ID NO: 19) IMP350: CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH₂

IMP350, incorporating the sequence of AD2, was made on a 0.1 mmol scalewith Sieber Amide resin using Fmoc methodology on a peptide synthesizer.Starting from the C-terminus the protected amino acids used wereFmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH andFmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified byreverse phase (RP)-HPLC.

Synthesis of PEG₂₀-IMP350

IMP350 (0.0104 g) was mixed with 0.1022 g of mPEG-OPTE (20 kDa, NEKTAR®Therapeutics) in 7 mL of 1 M Tris buffer at pH 7.81. Acetonitrile, 1 mL,was then added to dissolve some suspended material. The reaction wasstirred at room temperature for 3 h and then 0.0527 g of TCEP was addedalong with 0.0549 g of cysteine. The reaction mixture was stirred for1.5 h and then purified on a PD-10 desalting column, which wasequilibrated with 20% methanol in water. The sample was eluted, frozenand lyophilized to obtain 0.0924 g of crude PEG₂₀-IMP350 (MH+23508 byMALDI).

Synthesis of IMP362 (PEG₂₀-IMP360)

(SEQ ID NO: 20) IMP360: CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS MH⁺ 2660

IMP 360, incorporating the AD2 sequence, was synthesized on a 0.1 mmolscale with Fmoc-Gly-EDANS resin using Fmoc methodology on a peptidesynthesizer. The Fmoc-Gly-OH was added to the resin manually using 0.23g of Fmoc-Gly-OH, 0.29 g of HATU, 26 μL of DIEA, 7.5 mL of DMF and 0.57g of EDANS resin (NOVABIOCHEM®). The reagents were mixed and added tothe resin. The reaction was mixed at room temperature for 2.5 hr and theresin was washed with DMF and IPA to remove the excess reagents.Starting from the C-terminus the protected amino acids used wereFmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH andFmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified byRP-HPLC.

For synthesis of IMP362, IMP360 (0.0115 g) was mixed with 0.1272 g ofmPEG-OPTE (20 kDa, NEKTAR® Therapeutics) in 7 mL of 1 M tris buffer, pH7.81. Acetonitrile (1 mL) was then added to dissolve some suspendedmaterial. The reaction was stirred at room temperature for 4 h and then0.0410 g of TCEP was added along with 0.0431 g of cysteine. The reactionmixture was stirred for 1 h and purified on a PD-10 desalting column,which was equilibrated with 20% methanol in water. The sample waseluted, frozen and lyophilized to obtain 0.1471 g of crude IMP362(MH+23713).

Synthesis of IMP413 (PEG₃₀-IMP360)

For synthesis of IMP 413, IMP 360 (0.0103 g) was mixed with 0.1601 g ofmPEG-OPTE (30 kDa, NEKTAR® Therapeutics) in 7 mL of 1 M tris buffer atpH 7.81. Acetonitrile (1 mL) was then added to dissolve some suspendedmaterial. The reaction was stirred at room temperature for 4.5 h andthen 0.0423 g of TCEP was added along with 0.0473 g of cysteine. Thereaction mixture was stirred for 2 h followed by dialysis for two days.The dialyzed material was frozen and lyophilized to obtain 0.1552 g ofcrude IMP413 (MH⁺ 34499).

Synthesis of IMP421

IMP 421 (SEQ ID NO: 21)Ac-C-PEG₃-C(S-tBu)GQIEYLAKQIVDNAIQQAGC(S-tBu)G-NH₂

The peptide IMP421, MH⁺ 2891 was made on NOVASYN® TGR resin (487.6 mg,0.112 mmol) by adding the following amino acids to the resin in theorder shown: Fmoc-Gly-OH, Fmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH,Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH,Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Val-OH,Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH,Fmoc-Leu-OH, Fmoc-Tyr(But)-OH, Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Cys(t-Buthio)-OH, Fmoc-NH-PEG₃-COOH,Fmoc-Cys(Trt)-OH. The N-terminal amino acid was protected as an acetylderivative. The peptide was then cleaved from the resin and purified byRP-HPLC to yield 32.7 mg of a white solid.

Synthesis of IMP457

IMP 421 (SEQ ID NO:21), incorporating the sequence of AD2, wassynthesized by standard chemical means. To a solution of 15.2 mg (5.26μmol) IMP 421 (F.W. 2890.50) and 274.5 mg (6.86 μmol) mPEG2-MAL-40K in 1mL of acetonitrile was added 7 mL 1 M Tris pH 7.8 and allowed to reactat room temperature for 3 h. The excess mPEG2-MAL-40K was quenched with49.4 mg L-cysteine, followed by S—S-tBu deprotection over one hour with59.1 mg TCEP. The reaction mixture was dialyzed overnight at 2-8° C.using two 3-12 mL capacity 10K SLIDE-A-LYZER® dialysis cassettes (4 mlinto each cassette) into 5 L of 5 mM ammonium acetate, pH 5.0. Threemore 5 L buffer changes of 5 mM ammonium acetate, pH 5.0 were made thenext day with each dialysis lasting at least 2½ h. The purified product(19.4 mL) was transferred into two 20 mL scintillation vials, frozen andlyophilized to yield 246.7 mg of a white solid. MALDI-TOF gave resultsof mPEG2-MAL-40K 42,982 and IMP-457 45,500.

Example 14 Generation of PEGylated hMN-15 by DNL

A DNL structure is prepared having two copies of hMN-15 Fab coupled to a20 kDa PEG. A DNL reaction is performed by the addition of reduced andlyophilized IMP362 in 10-fold molar excess to hMN-15 Fab-DDD2 in 250 mMimidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH₂PO₄, pH7.5. After 6 h at room temperature in the dark, the reaction mixture isdialyzed against CM Loading Buffer (150 mM NaCl, 20 mM NaAc, pH 4.5) at4° C. in the dark. The solution is loaded onto a 1-mL Hi-Trap CM-FFcolumn (AMERSHAM®), which is pre-equilibrated with CM Loading buffer.After sample loading, the column is washed with CM loading buffer tobaseline, followed by washing with 15 mL of 0.25 M NaCl, 20 mM NaAc, pH4.5. The PEGylated hMN-15 is eluted with 12.5 mL of 0.5 M NaCl, 20 mMNaAc, pH 4.5.

The conjugation process is analyzed by SDS-PAGE with Coomassie bluestaining. Under non-reducing conditions, the Coomassie blue-stained gelreveals the presence of a major band in the reaction mixture, which isabsent in the unbound or 0.25 M NaCl wash fraction, but evident in the0.5 M NaCl fraction. Fluorescence imaging, which is used to detect theEDANS tag on IMP362, demonstrates that the band contains IMP362 and thepresence of excess IMP362 in the reaction mixture and the unboundfraction. The DNL reaction results in the site-specific and covalentconjugation of IMP362 with a dimer of hMN-15 Fab. Under reducingconditions, which breaks the disulfide linkage, the components of theDNL structures are resolved. The calculated MW of the (hMN-15 Fab)₂-PEGconstruct matches that determined by MALDI TOF. Overall, the DNLreaction results in a near quantitative yield of a homogeneous productthat is >90% pure after purification by cation-exchange chromatography.

Another DNL reaction is performed by the addition of reduced andlyophilized 1 MP457 in 10-fold molar excess to hMN-15 Fab-DDD2 in 250 mMimidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH₂PO₄, pH7.5. After 60 h at room temperature, 1 mM oxidized glutathione is addedto the reaction mixture, which is then held for an additional 2 h. Themixture is diluted 1:20 with CM Loading Buffer (150 mM NaCl, 20 mM NaAc,pH 4.5) and titrated to pH 4.5 with acetic acid. The solution is loadedonto a 1-mL Hi-Trap CM-FF column (AMERSHAM®), which is pre-equilibratedwith CM Loading Buffer. After sample loading, the column is washed withCM Loading Buffer to baseline, followed by washing with 15 mL of 0.25 MNaCl, 20 mM NaAc, pH 4.5. The PEGylated product is eluted with 20 mL of0.5 M NaCl, 20 mM NaAc, pH 4.5. The DNL construct is concentrated to 2mL and diafiltered into 0.4 M PBS, pH 7.4. The final PEGylated hMN-15Fab₂ construct is approximately 90% purity as determined by SDS-PAGE.

A DNL construct having two copies of hMN-15 Fab coupled to a 30 kDa PEGis prepared as described immediately above using IMP413 instead ofIMP362. The PEGylated hMN-15 Fab₂ DNL construct is purified as describedabove and obtained in approximately 90% purity. The PEGylated DNLconstructs may be used for therapeutic methods as described above fornon-PEGylated forms of hMN-15. It is observed that PEGylation of hMN-15Fab₂ increases the serum half-life of the hMN-15 moiety, as expected.

Example 15 Generation of DDD Module Based on Interferon (IFN)-α2b

The cDNA sequence for IFN-α2b was amplified by PCR, resulting in asequence comprising the following features, in which XbaI and BamHI arerestriction sites, the signal peptide is native to IFN-α2b, and 6 His isa hexahistidine tag (SEQ ID NO:37): XbaI—Signal peptide—IFNα2b—6His—BamHI (“6 His” disclosed as SEQ ID NO:37). The resulting secretedprotein consists of IFN-α2b fused at its C-terminus to a polypeptideconsisting of SEQ ID NO:22.

(SEQ ID NO: 22) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

PCR amplification was accomplished using a full length human IFNα2b cDNAclone (INVITROGEN® Ultimate ORF human clone cat#HORF01Clone ID IOH35221)as a template and the following oligonucleotides as primers:

IFNA2 Xba I Left (SEQ ID NO: 23)5′-TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACT GG-3′ IFNA2 BamHI right(SEQ ID NO: 24) 5′GGATCCATATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC-3′

The PCR amplimer was cloned into the PGEMT® vector (PROMEGA®). ADDD2-pdHL2 mammalian expression vector was prepared for ligation withIFN-α2b by digestion with XbaI and Bam HI restriction endonucleases. TheIFN-α2b amplimer was excised from PGEMT® with XbaI and Bam HI andligated into the DDD2-pdHL2 vector to generate the expression vectorIFN-α2b-DDD2-pdHL2.

IFN-α2b-DDD2-pdHL2 was linearized by digestion with Sail enzyme andstably transfected into Sp/EEE myeloma cells by electroporation (see,e.g., U.S. Pat. No. 7,537,930, the Examples section of which isincorporated herein by reference). Two clones were found to havedetectable levels of IFN-α2b by ELISA. One of the two clones, designated95, was adapted to growth in serum-free media without substantialdecrease in productivity. The clone was subsequently amplified withincreasing methotrexate (MTX) concentrations from 0.1 to 0.8 μM overfive weeks. At this stage, it was sub-cloned by limiting dilution andthe highest producing sub-clone (95-5) was expanded. The productivity of95-5 grown in shake-flasks was estimated to be 2.5 mg/L using commercialrIFN-α2b (CHEMICON® IF007, Lot 06008039084) as a standard.

Clone 95-5 was expanded to 34 roller bottles containing a total of 20 Lof serum-free Hybridoma SFM with 0.8 μM MTX and allowed to reachterminal culture. The supernatant fluid was clarified by centrifugationand filtered (0.2 μM). The filtrate was diafiltered into 1× Bindingbuffer (10 mM imidazole, 0.5 M NaCl, 50 mM NaH₂PO₄, pH 7.5) andconcentrated to 310 mL in preparation for purification by immobilizedmetal affinity chromatography (IMAC). The concentrate was loaded onto a30-mL Ni-NTA column, which was washed with 500 mL of 0.02% Tween 20 in1× binding buffer and then 290 mL of 30 mM imidazole, 0.02% Tween 20,0.5 M NaCl, 50 mM NaH₂PO₄, pH 7.5. The product was eluted with 110 mL of250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 50 mM NaH₂PO₄, pH 7.5.Approximately 6 mg of IFNα2b-DDD2 was purified.

The purity of IFN-α2b-DDD2 was assessed by SDS-PAGE under reducingconditions (not shown). IFN-α2b-DDD2 was the most heavily stained bandand accounted for approximately 50% of the total protein (not shown).The product resolved as a doublet with an M_(r) of ˜26 kDa, which isconsistent with the calculated MW of IFN-α2b-DDD2-SP (26 kDa). There wasone major contaminant with a M_(r) of 34 kDa and many faintcontaminating bands (not shown).

Example 16 Generation of hMN-15 Fab-(IFN-α2b)₂ by DNL

Creation of C—H-AD2-IgG-pdHL2 Expression Vectors.

The pdHL2 mammalian expression vector has been used to mediate theexpression of many recombinant IgGs. A plasmid shuttle vector wasproduced to facilitate the conversion of any IgG-pdHL2 vector into aC—H-AD2-IgG-pdHL2 vector. The gene for the Fc (CH2 and CH3 domains) wasamplified using the pdHL2 vector as a template and the oligonucleotidesFc BglII Left and Fc Bam-EcoRI Right as primers.

Fc BglII Left (SEQ ID NO: 25) 5′-AGATCTGGCGCACCTGAACTCCTG-3′Fc Bam-EcoRI Right (SEQ ID NO: 26)5′-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3′

The amplimer was cloned in the PGEMT® PCR cloning vector. The Fc insertfragment was excised from PGEMT® and ligated with AD2-pdHL2 vector togenerate the shuttle vector Fc-AD2-pdHL2.

Generation of hMN-15 IgG-AD2

To convert any IgG-pdHL2 expression vector to a C—H-AD2-IgG-pdHL2expression vector, an 861 by BsrGI/NdeI restriction fragment is excisedfrom the former and replaced with a 952 by BsrGI/NdeI restrictionfragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the CH3domain and NdeI cuts downstream (3′) of the expression cassette. Thismethod is used to generate an hMN-15 IgG-AD2 protein.

Generation of hMN-15 IgG-(IFN-α2b)₂ Construct

A DNL reaction is performed by the addition of reduced and lyophilizedhMN-15 IgG-AD2 to IFN-α2b-DDD2 in 250 mM imidazole, 0.02% Tween 20, 150mM NaCl, 1 mM EDTA, 50 mM NaH₂PO₄, pH 7.5. After 6 h at room temperaturein the dark, the reaction mixture is dialyzed against CM Loading Buffer(150 mM NaCl, 20 mM NaAc, pH 4.5) at 4° C. in the dark. The solution isloaded onto a 1-mL Hi-Trap CM-FF column (AMERSHAM®), which ispre-equilibrated with CM Loading buffer. After sample loading, thecolumn is washed with CM loading buffer to baseline, followed by washingwith 15 mL of 0.25 M NaCl, 20 mM NaAc, pH 4.5. The product is elutedwith 12.5 mL of 0.5 M NaCl, 20 mM NaAc, pH 4.5. The DNL reaction resultsin the site-specific and covalent conjugation of hMN-15 IgG with a dimerof IFN-α2b. Both the IgG and IFN-α2b moieties retain their respectivephysiological activities in the DNL construct. This technique may beused to attach any cytokine or other physiologically active protein orpeptide to hMN-15 for targeted delivery to colon cancer or other cancersthat express the CEA antigen.

Example 17 Preparation of DNL Bispecific Antibody Constructs

Methods of preparing bispecific DNL antibody constructs are described,for example, in U.S. Pat. No. 7,521,056, the methods section of which isincorporated herein by reference.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN14(I)-pdHL2, which has been used to produce hMN-14 IgG, was convertedto C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restrictionendonucleases to remove the CH1-CH3 domains and insertion of theCH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

Production of C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd via a 14amino acid residue Gly/Ser peptide linker. The fusion protein secretedis composed of two identical copies of hMN-14 Fab held together bynon-covalent interaction of the DDD2 domains.

The C-DDD2-Fd-hMN-14-pdHL2 vector was transfected into Sp/EEE myelomacells by electroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hMN-14 kappa light chain andC-DDD2-Fd-hMN-14, which combine to form C-DDD2-Fab-hMN14. Dimerizationoccurs via the DDD2 moiety, resulting in two reactive sulfhydryl groupsprovided by the cysteine residue in each DDD2. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 .mu.M methotrexate(MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with WI2 (hMN-14 anti-Id) and detection with goatanti-human Fab-HRP. The highest producing clones had an initialproductivity of approximately 100 mg/L. A total of 200 mg ofC-DDD2-hMN-14 was purified by protein L affinity chromatography from 1.8liters of roller bottle culture.

Generation of Trimeric hMN-14-hMN-15 Bispecific DNL Construct

hMN-15-Fab-AD2 is prepared as described in Example 12. hMN-14-Fab-DDD2is prepared as described above. The two fusion proteins (hMN-14-Fab-DDD2and hMN-15-Fab-AD2) are expressed independently in stably transfectedmyeloma cells. The tissue culture supernatant fluids are combined,resulting in a two-fold molar excess of hMN-14-Fab-DDD2. The reactionmixture is incubated at room temperature for 24 hours under mildreducing conditions using 1 mM reduced glutathione. Following reduction,the DNL reaction is completed by mild oxidation using 2 mM oxidizedglutathione. The trimeric bispecific DNL construct is isolated byaffinity chromatography using an anti-idiotypic antibody against hMN-14.The complex retains the binding affinities of both the hMN-14 and hMN-15antibodies.

Generation of Trimeric hPAM4-hMN-15 Bispecific DNL Construct

hMN-15-Fab-AD2 and hPAM4-Fab-DDD2 are prepared as described in Example11. The two fusion proteins (hPAM4-Fab-DDD2 and hMN-15-Fab-AD2) areexpressed independently in stably transfected myeloma cells. The tissueculture supernatant fluids are combined, resulting in a two-fold molarexcess of hPAM4-Fab-DDD2. The reaction mixture is incubated at roomtemperature for 24 hours under mild reducing conditions using 1 mMreduced glutathione. Following reduction, the DNL reaction is completedby mild oxidation using 2 mM oxidized glutathione. The trimericbispecific DNL construct is isolated by affinity chromatography using ananti-idiotypic antibody against hMN-15. The complex retains the bindingaffinities of both the hPAM4 and hMN-15 antibodies.

The skilled artisan will realize that a trimeric Fab construct can beprepared using virtually any antibodies that have been cloned in anexpression vector, using appropriate restriction endonucleases andstandard molecular cloning techniques. Thus, combinations of hMN-15 withany other antibody or antibody fragment may be prepared by DNL.

Example 18 Production of Targeting Peptides for Use in Pretargeting and¹⁸F Labeling

In certain embodiments, ¹⁸F-labeled proteins or peptides are prepared bya novel technique and used for diagnostic and/or imaging studies, suchas PET imaging. The novel technique for ¹⁸F labeling involvespreparation of an ¹⁸F-metal complex, preferably an ¹⁸F-aluminum complex,which is chelated to a chelating moiety, such as DOTA, NOTA or NETA orderivatives thereof. Chelating moieties may be attached to proteins,peptides or any other molecule using conjugation techniques well knownin the art. In certain preferred embodiments, the ¹⁸F—Al complex isformed in solution first and then attached to a chelating moiety that isalready conjugated to a protein or peptide. However, in alternativeembodiments the aluminum may be first attached to the chelating moietyand the ¹⁸F added later.

Peptide Synthesis

Peptides were synthesized by solid phase peptide synthesis using theFmoc strategy. Groups were added to the side chains of diamino aminoacids by using Fmoc/Aloc protecting groups to allow differentialdeprotection. The Aloc groups were removed by the method of Dangles et.al. (J. Org. Chem. 1987, 52:4984-4993) except that piperidine was addedin a 1:1 ratio to the acetic acid used. The unsymmetrical tetra-t-butylDTPA was made as described in McBride et al. (U.S. Pat. No. 7,405,320,the Examples section of which is incorporated herein by reference).

The tri-t-butyl DOTA, symmetrical tetra-t-butyl DTPA, ITC-benzyl DTPA,p-SCN-Bn-NOTA and TACN were obtained from MACROCYCLICS® (Dallas, Tex.).The DiBocTACN, NODA-GA(tBu)₃ and the NO2AtBu were purchased fromCheMatech (Dijon, France). The Aloc/Fmoc Lysine and Dap(diaminopropionic acid derivatives (also Dpr)) were obtained fromCREOSALUS® (Louisville, Ky.) or BACHEM® (Torrance, Calif.). The SieberAmide resin was obtained from NOVABIOCHEM® (San Diego, Calif.). Theremaining Fmoc amino acids were obtained from CREOSALUS®, BACHEM®,PEPTECH® (Burlington, Mass.), EMD BIOSCIENCES® (San Diego, Calif.), CHEMIMPEX® (Wood Dale, Ill.) or NOVABIOCHEM®. The aluminum chloridehexahydrate was purchased from SIGMA-ALDRICH® (Milwaukee, Wis.). Theremaining solvents and reagents were purchased from FISHER SCIENTIFIC®(Pittsburgh, Pa.) or SIGMA-ALDRICH® (Milwaukee, Wis.). ¹⁸F was suppliedby IBA MOLECULAR®(Somerset, N.J.)

¹⁸F-Labeling of IMP 272

The first peptide that was prepared and ¹⁸F-labeled was IMP 272:

DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂ MH⁺ 1512

IMP 272 was synthesized as described (McBride et al., U.S. Pat. No.7,534,431, the Examples section of which is incorporated herein byreference).

Acetate buffer solution—Acetic acid, 1.509 g was diluted in ˜160 mLwater and the pH was adjusted by the addition of 1 M NaOH then dilutedto 250 mL to make a 0.1 M solution at pH 4.03.

Aluminum acetate buffer solution—A solution of aluminum was prepared bydissolving 0.1028 g of AlCl₃ hexahydrate in 42.6 mL DI water. A 4 mLaliquot of the aluminum solution was mixed with 16 mL of a 0.1 M NaOAcsolution at pH 4 to provide a 2 mM Al stock solution.

IMP 272 acetate buffer solution—Peptide, 0.0011 g, 7.28×10⁻⁷ mol IMP 272was dissolved in 364 μL of the 0.1 M pH 4 acetate buffer solution toobtain a 2 mM stock solution of the peptide.

F-18 Labeling of IMP 272—A 3 μL aliquot of the aluminum stock solutionwas placed in a REACTI-VIALT™ and mixed with 50 μL ¹⁸F (as received) and3 μL of the IMP 272 solution. The solution was heated in a heating blockat 110° C. for 15 min and analyzed by reverse phase HPLC. HPLC analysis(not shown) showed 93% free ¹⁸F and 7% bound to the peptide. Anadditional 10 μL of the IMP 272 solution was added to the reaction andit was heated again and analyzed by reverse phase HPLC (not shown). TheHPLC trace showed 8% ¹⁸F at the void volume and 92% of the activityattached to the peptide. The remainder of the peptide solution wasincubated at room temperature with 150 μL PBS for ˜1 hr and thenexamined by reverse phase HPLC. The HPLC (not shown) showed 58% ¹⁸Funbound and 42% still attached to the peptide. The data indicate that¹⁸F—Al-DTPA complex may be unstable when mixed with phosphate.

The labeled peptide was purified by applying the labeled peptidesolution onto a 1 cc (30 mg) WATERS® HLB column (Part #186001879) andwashing with 300 μL water to remove unbound F-18. The peptide was elutedby washing the column with 2×100 μL 1:1 EtOH/H₂O. The purified peptidewas incubated in water at 25° C. and analyzed by reverse phase HPLC (notshown). The HPLC analysis showed that the ¹⁸F-labeled IMP 272 was notstable in water. After 40 min incubation in water about 17% of the ¹⁸Fwas released from the peptide, while 83% was retained (not shown).

The peptide (16 μL 2 mM IMP 272, 48 μg) was labeled with ¹⁸F andanalyzed for antibody binding by size exclusion HPLC. The size exclusionHPLC showed that the peptide bound hMN-14×679 but did not bind to theirrelevant bispecific antibody hMN-14×734 (not shown).

IMP 272¹⁸F Labeling with Other Metals

A ˜3 μL aliquot of the metal stock solution (6×10 ^(g) mol) was placedin a polypropylene cone vial and mixed with 75 μL ¹⁸F (as received),incubated at room temperature for ˜2 min and then mixed with 20 μL of a2 mM (4×10⁻⁸ mol) IMP 272 solution in 0.1 M NaOAc pH 4 buffer. Thesolution was heated in a heating block at 100° C. for 15 min andanalyzed by reverse phase HPLC. IMP 272 was labeled with indium (24%),gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (notshown). These results demonstrate that the ¹⁸F metal labeling techniqueis not limited to an aluminum ligand, but can also utilize other metalsas well. With different metal ligands, different chelating moieties maybe utilized to optimize binding of an F-18-metal conjugate.

Production and Use of a Serum-Stable ¹⁸F-Labeled Peptide IMP 449

The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ MH⁺ 1009 wasmade on Sieber Amide resin by adding the following amino acids to theresin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc wascleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Alocwas cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to make the desiredpeptide. The peptide was then cleaved from the resin and purified byHPLC to produce IMP 448, which was then coupled to ITC-benzyl NOTA. Thepeptide, IMP 448, 0.0757 g (7.5×10⁻⁵ mol) was mixed with 0.0509 g(9.09×10⁻⁵ mol) ITC benzyl NOTA and dissolved in 1 mL water. Potassiumcarbonate anhydrous (0.2171 g) was then slowly added to the stirredpeptide/NOTA solution. The reaction solution was pH 10.6 after theaddition of all the carbonate. The reaction was allowed to stir at roomtemperature overnight. The reaction was carefully quenched with 1 M HClafter 14 hr and purified by HPLC to obtain 48 mg of IMP 449.

¹⁸F Labeling of IMP 449

The peptide IMP 449 (0.002 g, 1.37×10⁻⁶ mol) was dissolved in 686 μL (2mM peptide solution) 0.1 M NaOAc pH 4.02. Three microliters of a 2 mMsolution of Al in a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi of¹⁸F. The solution was then mixed with 20 μL of the 2 mM IMP 449 solutionand heated at 105° C. for 15 min. Reverse phase HPLC analysis showed 35%(t_(R)˜10 min) of the activity was attached to the peptide and 65% ofthe activity was eluted at the void volume of the column (3.1 min, notshown) indicating that the majority of activity was not associated withthe peptide. The crude labeled mixture (5 μL) was mixed with pooledhuman serum and incubated at 37° C. An aliquot was removed after 15 minand analyzed by HPLC. The HPLC showed 9.8% of the activity was stillattached to the peptide (down from 35%). Another aliquot was removedafter 1 hr and analyzed by HPLC. The HPLC showed 7.6% of the activitywas still attached to the peptide (down from 35%), which was essentiallythe same as the 15 min trace (data not shown).

High Dose ¹⁸F Labeling

Further studies with purified IMP 449 demonstrated that the ¹⁸F-labeledpeptide was highly stable (91%, not shown) in human serum at 37° C. forat least one hour and was partially stable (76%, not shown) in humanserum at 37° C. for at least four hours. Additional studies wereperformed in which the IMP 449 was prepared in the presence of ascorbicacid as a stabilizing agent. In those studies (not shown), themetal-¹⁸F-peptide complex showed no detectable decomposition in serumafter 4 hr at 37° C. The mouse urine 30 min after injection of¹⁸F-labeled peptide was found to contain ¹⁸F bound to the peptide (notshown). These results demonstrate that the ¹⁸F-labeled peptidesdisclosed herein exhibit sufficient stability under approximated in vivoconditions to be used for ¹⁸F imaging studies.

For studies in the absence of ascorbic acid, ¹⁸F ˜21 mCi in ˜400 μL ofwater was mixed with 9 μL of 2 mM AlCl₃ in 0.1 M pH 4 NaOAc. Thepeptide, IMP 449, 60 μL (0.01 M, 6×10⁻⁷ mol in 0.5 NaOH pH 4.13) wasadded and the solution was heated to 110° C. for 15 min. The crudelabeled peptide was then purified by placing the reaction solution inthe barrel of a 1 cc WATERS® HLB column and eluting with water to removeunbound ¹⁸F followed by 1:1 EtOH/H₂0 to elute the ¹⁸F-labeled peptide.The crude reaction solution was pulled through the column into a wastevial and the column was washed with 3×1 mL fractions of water (18.97mCi). The HLB column was then placed on a new vial and eluted with 2×200μL 1:1 EtOH/H₂O to collect the labeled peptide (1.83 mCi). The columnretained 0.1 mCi of activity after all of the elutions were complete. Analiquot of the purified ¹⁸F-labeled peptide (20 μL) was mixed with 200μL of pooled human serum and heated at 37° C. Aliquots were analyzed byreverse phase HPLC. The results showed the relative stability of¹⁸F-labeled purified IMP 449 at 37° C. at time zero, one hour (91%labeled peptide), two hours (77% labeled peptide) and four hours (76%labeled peptide) of incubation in human serum (not shown). It was alsoobserved that ¹⁸F-labeled IMP 449 was stable in TFA solution, which isoccasionally used during reverse phase HPLC chromatography. Thereappears to be a general correlation between stability in TFA andstability in human serum observed for the exemplary ¹⁸F-labeledmolecules described herein. These results demonstrate that ¹⁸F-labeledpeptide, produced according to the methods disclosed herein, showssufficient stability in human serum to be successfully used for in vivolabeling and imaging studies, for example using PET scanning to detectlabeled cells or tissues. Finally, since IMP 449 peptide contains athiourea linkage, which is sensitive to radiolysis, several products areobserved by RP-HPLC. However, when ascorbic acid is added to thereaction mixture, the side products generated were markedly reduced.

Example 19 In Vivo Studies with Pretargeting Using MN-15-679 DNLConstruct and ¹⁸F-Labeled Peptide

¹⁸F-labeled IMP 449 is prepared as follows. The ¹⁸F, 54.7 mCi in ˜0.5 mLis mixed with 3 μL 2 mM Al in 0.1 M NaOAc pH 4 buffer. After 3 min 10 μLof 0.05 M IMP 449 in 0.5 M pH 4 NaOAc buffer is added and the reactionis heated in a 96° C. heating block for 15 min. The contents of thereaction are removed with a syringe. The crude labeled peptide is thenpurified by HPLC on a C₁₈ column. The flow rate is 3 mL/min. Buffer A is0.1% TFA in water and Buffer B is 90% acetonitrile in water with 0.1%TFA. The gradient goes from 100% A to 75/25 A:B over 15 min. There isabout 1 min difference in retention time (t_(R)) between the labeledpeptide, which eluted first and the unlabeled peptide. The HPLC eluentis collected in 0.5 min (mL) fractions. The labeled peptide has a t_(R)between 6 to 9 min depending on the column used. The HPLC purifiedpeptide sample is further processed by diluting the fractions ofinterest two fold in water and placing the solution in the barrel of a 1cc WATERS® HLB column. The cartridge is eluted with 3×1 mL water toremove acetonitrile and TFA followed by 400 μL 1:1 EtOH/H₂O to elute the¹⁸F-labeled peptide. The purified [Al¹⁸F] IMP 449 elutes as a singlepeak on an analytical HPLC C₁₈ column (not shown).

Female athymic mice (nu/nu) bearing GW-39 xenografts are used for invivo studies. Three of the mice are injected with hMN-15-679 DNLconstruct (162 μg) followed with [Al¹⁸F] IMP 449 18 h later. ThehMN-15-679 DNL construct is a humanized bispecific antibody of use fortumor imaging studies, with divalent binding to the CEA tumor antigenand monovalent binding to HSG. One mouse is injected with peptide alone.All of the mice are necropsied at 1 h post peptide injection. Tissuesare counted immediately. Comparison of mean distributions showssubstantially higher levels of ¹⁸F-labeled peptide localized in thetumor than in any normal tissues in the presence of tumor-targetingbispecific antibody. The results demonstrate that ¹⁸F labeled peptideused in conjunction with an hMN-15 containing antibody construct, suchthe hMN-15-679 DNL construct, provide suitable targeting of the ¹⁸Flabel to perform in vivo imaging, such as PET imaging analysis.

Example 20 AD and DDD Sequence Variants

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the DNL complexes comprise the amino acid sequences of AD2 (SEQ IDNO:14) and DDD2 (SEQ ID NO:12), as described above. However, inalternative embodiments sequence variants of the AD and/or DDD moietiesmay be utilized in construction of the cytokine-MAb DNL complexes. Thestructure-function relationships of the AD and DDD domains have been thesubject of investigation. (See, e.g., Burns-Hamuro et al., 2005, ProteinSci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto etal., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006,Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Goldet al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:11below. (See FIG. 1 of Kinderman et al., 2006.) The skilled artisan willrealize that in designing sequence variants of the DDD sequence, onewould desirably avoid changing any of the underlined residues, whileconservative amino acid substitutions might be made for residues thatare less critical for dimerization and AKAP binding.

Human DDD sequence from protein kinase A (SEQ ID NO: 11)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (NO:13), with a binding constant for DDD of 0.4 nM. The AKAP-ISsequence was designed as a peptide antagonist of AKAP binding to PKA.Residues in the AKAP-IS sequence where substitutions tended to decreasebinding to DDD are underlined in SEQ ID NO:13 below.

AKAP-IS sequence (SEQ ID NO: 13) QIEYLAKQIVDNAIQQA

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:27), exhibiting a five orderof magnitude higher selectivity for the RH isoform of PKA compared withthe R1 isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, which increased bindingto the DDD moiety of RIM. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIM, were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare cytokine-MAb DNL constructs. Other alternativesequences that might be substituted for the AKAP-IS AD sequence areshown in SEQ ID NO:28-30. Substitutions relative to the AKAP-IS sequenceare underlined. It is anticipated that, as with the AKAP-IS sequenceshown in SEQ ID NO:14, the AD moiety may also include the additionalN-terminal residues cysteine and glycine and C-terminal residues glycineand cysteine.

SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 27)Alternative AKAP sequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 28)QIEYHAKQIVDHAIHQA (SEQ ID NO: 29) QIEYVAKQIVDHAIHQA (SEQ ID NO: 30)

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:31-33. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:31), RIAD (SEQ ID NO:32) and PV-38 (SEQ IDNO:33). The Ht-31 peptide exhibited a greater affinity for the RHisoform of PKA, while the RIAD and PV-38 showed higher affinity for R1.

Ht31 (SEQ ID NO: 31) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 32)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 33) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:13). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.) The sequences of peptide antagonistswith particularly high affinities for the RII DDD sequence are shown inSEQ ID NO:34-36.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 13) AKA7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 34) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 35) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 36)

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:11. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins.

(SEQ ID NO: 11) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR LREA R A

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

The skilled artisan will realize that these and other amino acidsubstitutions in the antibody moiety or linker portions of the DNLconstructs may be utilized to enhance the therapeutic and/orpharmacokinetic properties of the resulting DNL constructs.

Example 21 Reactivity of MN-3 and MN-15 with CEA, NCA-90 and NCA-95 byIndirect Flow Cytometry

The reacitivty of antibodies was tested by flow cytometry with aBecton-Dickinson FACSCAN®. The biotinylated second antibody was directedagainst mouse IgG and was detected by a streptavidin/phycoerythrinconjugate. Tests were carried out with antibodies at 1/50 dilution of 1mg/ml stocks.

The antibodies MN-3 and MN-15 were tested for recognition of HeLa cellstransfected with cDNA coding for CEA (HeLa-CEA2), NCA-90 (HeLa-KNC6/S44)and NCA-95 (HeLa-CGM6/1). As a negative control, HeLa cells transfectedwith the plasmid pSV2-Neo (control HeLa cells, HeLa-NeoA) were used. Inaddition to the antibodies MN-3 and MN-15, three control antibodies wereincluded:

MAb 47 which cross reacts with CEA and NCA-95, but not NCA-90

MAb A which reacts with CEA

MAb NA which reacts with NCA-90

The results of the analysis by indirect fluorescence flow cytometryshowed that MN-3 binds NCA-90 and also CEA, but not NCA-95 (not shown).In contrast, MN-15 binds to all three antigens—CEA, NCA-90 and NCA-95(not shown). Control antibodies included in the panel tested showed theexpected cross-reactivities, as predicted from the literature (Berlinget al., Cancer Res 1990, 50:6534-39).

1. A method of treating a disease selected from the group consisting ofinfection, inflammation and sepsis, comprising administering to a humansubject a chimeric, humanized or human Class I anti-CEA antibody orfragment thereof, wherein the Class I anti-CEA antibody or fragmentthereof comprises the light chain variable region CDR sequencesSASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ IDNO:3); and the heavy chain variable region CDR sequences DYYMS (SEQ IDNO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6).2. The method of claim 1, wherein the Class I anti-CEA antibody orfragment thereof is a naked antibody or fragment thereof.
 3. The methodof claim 2, further comprising administering at least one therapeuticagent to the subject.
 4. The method of claim 3, wherein the therapeuticagent is selected from the group consisting of a naked antibody, anantibody fragment, a cytotoxic agent, a drug, a radionuclide, boronatoms, an immunomodulator, a photoactive therapeutic agent, animmunoconjugate, an oligonucleotide and a hormone.
 5. The method ofclaim 1, wherein the Class I anti-CEA antibody or fragment thereof isconjugated to at least one therapeutic agent.
 6. The method of claim 5,wherein the therapeutic agent is selected from the group consisting ofan antibody, an antibody fragment, a cytotoxic agent, a drug, aradionuclide, boron atoms, an immunomodulator, a photoactive therapeuticagent, an immunoconjugate, an oligonucleotide and a hormone.
 7. Themethod of claim 6, wherein the cytotoxic agent is a drug or toxin. 8.The method of claim 7, wherein the drug has a pharmaceutical propertyselected from the group consisting of antimitotic, alkylating,antimetabolite, antiangiogenic, apoptotic, alkaloid, COX-2, andantibiotic agents.
 9. The method of claim 7, wherein the drug isselected from the group consisting of nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs,purine analogs, antimetabolites, antibiotics, enzymes,epipodophyllotoxins, platinum coordination complexes, vinca alkaloids,substituted ureas, methyl hydrazine derivatives, adrenocorticalsuppressants, antagonists, endostatin, taxols, camptothecins,oxaliplatin and doxorubicins.
 10. The method of claim 7, wherein thedrug is selected from the group consisting of 5-fluorouracil, aplidin,azaribine, anastrozole, anthracyclines, bendamustine, bleomycin,bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin,carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil,cisplatin (CDDP), COX-2 inhibitors, irinotecan (CPT-11), SN-38,carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine,dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicin (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosurea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vinorelbine, vinblastine, vincristine andvinca alkaloids.
 11. The method of claim 7, wherein the toxin isselected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin and Pseudomonas endotoxin.
 12. The method of claim6, wherein the oligonucleotide is selected from the group consisting ofan RNAi and a siRNA.
 13. The method of claim 6, wherein the radionuclideis selected from the group consisting of ¹¹¹In, ¹¹¹At, ¹⁷⁷Lu, ²¹¹Bi,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹³³I, ³²P, ³³P, ⁴⁷Sc,¹¹¹Ag, ⁶⁷Ga, ¹⁵³Sm, ¹⁶¹Tb, ¹⁵²Dy, ¹⁶⁶Dy, ¹⁶¹Ho, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ²¹¹Pb, ²¹²Pb, ²²³Ra, ²²⁵Ac, A ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ⁵⁸Co, ^(80m)Br, ^(99m)Tc, ^(103m)Rh, ¹⁰⁹Pt, ¹¹⁹Sb, ¹²⁵I,^(189m)Os, ¹⁹²Ir, ²¹⁹Rn, ²¹⁵Po, ²²¹Fr, ²⁵⁵Fm, ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ¹⁹⁹Au, ²²⁴Ac, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg,²⁰³Hg, ^(121m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁴²Pr,¹⁴³Pr, ¹⁶¹Tb, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ⁷⁶Br and ¹⁶⁹Yb, 14.The method of claim 6, wherein the immunomodulator is selected from thegroup consisting of a cytokine, a chemokine, a stem cell growth factor,a lymphotoxin, an hematopoietic factor, a colony stimulating factor(CSF), an interferon, erythropoietin, thrombopoietin, a tumor necrosisfactor (TNF), an interleukin (IL), granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF) and astem cell growth factor designated “S1 factor”.
 15. The method of claim14, wherein the cytokine is selected from the group consisting of humangrowth hormone, N-methionyl human growth hormone, bovine growth hormone,parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, tumor necrosis factor-α, tumor necrosis factor-β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,erythropoietin (EPO), osteoinductive factors, interferon-α,interferon-β, interferon-γ, macrophage-CSF (M-CSF), IL-1, IL-1αIL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3,angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.16. The method of claim 14, wherein the chemokine is selected from thegroup consisting of RANTES, MCAF, MIP1-alpha, MIP1-beta and IT-10. 17.The method of claim 1, wherein the chimeric, humanized or human Class Ianti-CEA antibody or fragment is part of a bispecific antibody furthercomprising a second antibody or fragment thereof that binds to atargetable construct and the method further comprises administering atargetable construct to the subject, wherein the targetable construct isconjugated to at least one therapeutic agent.
 18. The method of claim17, wherein the therapeutic agent is selected from the group consistingof an antibody, an antibody fragment, a cytotoxic agent, a drug, aradionuclide, boron atoms, an immunomodulator, a photoactive therapeuticagent, an immunoconjugate, an oligonucleotide and a hormone.
 19. Themethod of claim 1 wherein the chimeric, humanized or human Class Ianti-CEA antibody or fragment is a fusion protein.
 20. The method ofclaim 1, wherein the Class I anti-CEA antibody or fragment thereofcomprises human antibody IgG1 constant region sequences.
 21. The methodof claim 1, wherein the Class I anti-CEA antibody or fragment thereof isadministered as a pharmaceutical composition.
 22. The method of claim 1,wherein the Class I anti-CEA antibody or fragment thereof is a chimericantibody or fragment thereof.
 23. The method of claim 22, wherein thechimeric Class I anti-CEA antibody or fragment thereof comprises thevariable region amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10.24. The method of claim 1, wherein Class I anti-CEA antibody or fragmentthereof is a humanized antibody or fragment thereof.
 25. The method ofclaim 24, wherein the humanized Class I anti-CEA antibody or fragmentthereof comprises the amino acid sequences of SEQ ID NO:7 and SEQ IDNO:8.
 26. The method of claim 24, wherein the humanized Class I anti-CEAantibody or fragment thereof comprises the light chain FR sequences of ahuman REI antibody and the heavy chain FR sequences of a human KOLantibody.
 27. The method of claim 24, wherein the framework regions ofthe light and heavy chain variable regions of the humanized antibody orfragment thereof comprise at least one amino acid substitution selectedfrom the heavy chain amino acid residues 28, 29, 30, 48 and 49 of SEQ IDNO:10 and the light chain amino acid residues 21, 47 and 60 of SEQ IDNO:9.
 28. The method of claim 1, wherein the antibody fragment isselected from the group consisting of F(ab′)₂, Fab′, Fab, Fv and scFv.29. The method of claim 1, wherein the disease is an infection.
 30. Themethod of claim 1, wherein the disease is an inflammation.
 31. Themethod of claim 1, wherein the disease is sepsis.